Methods for controlling SR protein phosphorylation, and antiviral agents whose active ingredients comprise agents that control SR protein activity

ABSTRACT

The present invention provides: (1) antiviral agents that act by reducing or inhibiting the activity of SR proteins, more specifically, (i) antiviral agents that act by enhancing dephosphorylation of SR proteins, and (ii) antiviral agents that act by inhibiting proteins that phosphorylate SR proteins; (2) antiviral agents that act by inhibiting the expression of SR proteins, and (3) antiviral agents that act by activating proteins that antagonize SR proteins. The present invention also provides compounds that inhibit SRPKs, which phosphorylate SR proteins. Such compounds inhibit the activity of SR proteins and have antiviral activities. Various new viruses including SARS have emerged, and thus the present invention provides long-lasting broad-spectrum antiviral agents applicable to new viruses.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.12/494,102, filed Jun. 29, 2009, which is a continuation of U.S. patentapplication Ser. No. 10/584,482, filed Mar. 2, 2007, now U.S. Pat. No.7,569,536, which is the §371 U.S. National Stage of InternationalApplication No. PCT/JP2004/019393, filed Dec. 24, 2004, which in turnclaims the benefit of Japan Application No. 2003-435085, filed Dec. 26,2003. The disclosures of U.S. patent application Ser. Nos. 12/494,102and 10/584,482 are incorporated by reference herein in their entirety.

TECHNICAL FIELD

The present invention relates to controlling the phosphorylation of SRproteins, which are involved in splicing reactions in the process ofgene expression. The present invention also relates to methods forcontrolling the activity, expression, and stabilization of SR proteinsthat are useful for treating and preventing chronic and acute diseasescaused by the infection of viruses or such, and antiviral agents whoseactive ingredients comprise agents that control SR protein activity.Furthermore, the present invention also relates to compounds useful forcontrolling SR protein activity and for antiviral treatments, and usesthereof.

BACKGROUND ART

Many of the antiviral agents reported to date as inhibiting viralreplication are targeted at viral proteases or reverse transcriptases ofviruses and so on.

For example, in the case of HIV virus, methods targeting thecharacteristics of the HIV genome are used. HIV's RNA genome isconverted into DNA (provirus) by reverse transcriptase, and is thenintegrated into host chromosomes. Then, the transcription andtranslation mechanisms of the host cells produce viral proteins from theproviral DNA. These proteins are expressed as large polyproteinprecursors. The precursors are cleaved into proteins by proteases, andthen HIV virus is re-constituted and matured. Thus, HIV inhibitorstargeted to each step in this HIV maturation process have been studiedand developed; such inhibitors include (1) AZT and the like, which aretargeted at reverse transcriptases characteristic of retroviruses(Non-patent Document 1) and (2) protease inhibitors, which inhibitproteases (Non-patent Document 2).

However, all of these are individually targeted antiviral agents thatspecifically attack the propagation process of the various viruses.

-   Non-patent Document 1: Proc Natl Acad Sci USA Vol. 83, No. 21, pp.    8333-7-   Non-patent Document 2: Antimicrob Agents Chemother. 1995 July;    39(7):1559-64    Sequence Listing

The nucleic and amino acid sequences provided herein are shown usingstandard letter abbreviations for nucleotide bases, and three lettercodes for amino acids, as defined in 37 C.F.R. 1.822. Only one strand ofeach nucleic acid sequence is shown, but the complementary strand isunderstood as included by any reference to the displayed strand. TheSequence Listing is submitted as an ASCII text file in the form of thefile named “Sequence.txt” (˜32 kb), which was created on Nov. 16, 2012,which is incorporated by reference herein.

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention

Since the natural rate of mutation is higher in viruses, and in RNAviruses in particular, the antiviral agents developed thus far, whichtarget viral proteases or reverse transcriptases of viruses and so on,often rapidly lose their efficacy. Thus, the development of moreeffective antiviral agents is desired.

Specifically, in accordance with the recent emergence of various newviruses, including SARS, an objective of the present invention is todevelop long-lasting broad-spectrum antiviral agents that are alsoapplicable to new viruses.

Means to Solve the Problems

The present inventors previously studied the phosphorylation of SRproteins, which are involved in gene expression systems. In particular,the present inventors were the first in the world to clone SRPK2, anenzyme that phosphorylates SR proteins (Biochem. Biophys. Res. Commun.242, 357-364), SPK1, a nematode SRPK homolog (Mech. Dev. 99, 51-64),hPRP4 (J. Biol. Chem. 277, 44220-44228), and CLASP, a regulatory factorfor SR protein kinase Clk4 (J. Biol. Chem. 276, 32247-32256).

SR proteins are RNA-binding proteins rich in serine and arginine. SRproteins typically share one or two RNA-recognition motifs (RRM) and anRS (Arginine/Serine-rich) domain that is rich in consecutive RSsequences. The proteins play an important role in eukaryotic RNAprocessing, and in splicing of pre-mRNA in particular.

The following ten types of RNA-binding proteins belonging to themammalian SR protein family have been reported: X16/SRp20,SF2/ASF/SRp30a, SC35/PR264/SRp30b, SRp30c, 9G8, HRS/SRp40, SRp46, SRp55,SRp75, and p54. Most SR proteins have been shown to be phosphorylated incells. In particular, peptide mapping analysis has revealed thatSF2/ASF, an SR protein, is phosphorylated at multiple sites within theRS domain (J. Cell. Biol. (1991) 115: 587-596). Furthermore, thephosphorylation is known to potentiate the ability of SF2/ASF toselectively bind to U1snRNP (Genes & Dev. (1997) 11: 334-344). Thephosphorylation and dephosphorylation of RS domains is required forspliceosome formation and rearrangement. When this phosphorylation anddephosphorylation is inhibited, mRNA processing becomes abnormal. RSdomains are found not only in RNA-binding proteins, as described above,but also in various functional proteins thought to function in thenucleus. These proteins have been named the “SR-related protein family”(Biochem. Cell Biol. (1999) 77: 277-291).

When studying the phosphorylation states of SR family proteins invirus-infected cells, the present inventors unexpectedly discovered thatin virus-infected cells phosphorylation of SR proteins was inhibited,and these proteins were degraded via the ubiquitin-proteasome pathway.They also discovered a phenomenon whereby, conversely, SR proteins werestabilized and virus production increased upon forced expression of anSR protein, such as SRp40 or SRp75, or an SR protein kinase, such asSRPK1 or SRPK2. This suggests that SR proteins play an important role inviral replication, and that the dephosphorylation of SR proteinsfunctions as a defense system against viral invasion of the body.

SR proteins bind to U1snRNP or U2AF, and are required for spliceosomeformation; the RS domains are thought to play a major role in thatprotein-protein interaction. Furthermore, SR proteins influence splicesite selection, promoting the selection of 3′ splice sites proximal toan intron. In contrast, heteronuclear ribonucleoproteins (hnRNPs), suchas hnRNP A1, A2, and B1, promote the selection of distal 3′ splicesites. Thus, splice site selection may depend on the intracellular ratioof SR protein and hnRNP protein.

The present inventors thus developed and provided antiviral agentstargeting SR proteins, which were found to play an important role inviral replication.

Specifically, first, the inventors attempted to inhibit SR proteins byinhibiting SR protein kinase.

Since there were no known low-molecular-weight compounds that inhibitedSRPK activity, the present inventors screened for low-molecular-weightcompounds targeting SRPK. As a result, they discovered that SRPIN-1 (SRprotein phosphorylation inhibitor 1; also referred to as Compound No.340) had the activity of inhibiting the kinase, SRPK; SRPIN-1 isrepresented by the following formula:

The present inventors thus speculated that viral replication of HIVcould be inhibited as a result of inhibiting SR protein phosphorylationby using SRPIN-1 to inhibit the enzymatic activity of SRPKs. Usingvarious concentrations of SRPIN-1, they tested whether viral replicationcould be inhibited in infection experiments using MT-4 cells and HIV.They thus discovered that SRPIN-1 markedly inhibited HIV replication.

Further, the present inventors synthesized multiple SRPIN-1 analogs andtested their effect. Like SRPIN-1, the analogs were found to showSRPK-inhibiting activity and antiviral activity. Thus, SRPIN-1 andanalogs thereof are useful as SRPK inhibitors, and can also be used asantiviral agents.

Specifically, the present invention relates to antiviral agents whoseactive ingredients comprise SR activity-controlling agents that controlSR protein activity, methods for screening for antiviral agents,compounds with the activity of inhibiting SRPK, uses thereof, and such.More specifically, the present invention relates to each claim of thepresent invention:

-   [1] an antiviral agent comprising as an active ingredient an SR    activity-controlling agent that controls an activity of an SR    protein;-   [2] the antiviral agent of [1], wherein the SR protein is any one of    SF2/ASF/SRp30a, SC35/PR264/SRp30b, SRp30c, HRS/SRp40, SRp46, or    SRp75;-   [3] the antiviral agent of [1] or [2], wherein the SR    activity-controlling agent is a substance or composition that    enhances dephosphorylation of an SR protein;-   [4] the antiviral agent of [3], which is an activator that activates    Phosphatase 2A;-   [5] the antiviral agent of [4], which is an expression vector for    gene therapy, which carries an HIV tat gene, an adenovirus E4-ORF4    gene, or a vaccinia virus VH1 gene;-   [6] the antiviral agent of [1] or [2], wherein the SR    activity-controlling agent is a substance that inhibits an SRPK;-   [7] the antiviral agent of [6], wherein the SRPK is an SRPK 1 or    SRPK 2;-   [8] the antiviral agent of [1] or [2], wherein the SR    activity-controlling agent is an SRPK gene expression inhibitor;-   [9] the antiviral agent of [8], wherein the SRPK gene expression    inhibitor is an miRNA, siRNA, or morpholino oligo targeting an SRPK,    or an expression vector for the miRNA or siRNA;-   [10] the antiviral agent of [1] or [2], wherein the SR    activity-controlling agent is a substance having the activity of    antagonizing an SR protein;-   [11] the antiviral agent of [10], wherein the substance having the    activity of antagonizing an SR protein is an expression vector for    hnRNPA1;-   [12] the antiviral agent of any one of [1] to [11], wherein the    virus is: (1) any one of the following RNA viruses: a human    immunodeficiency virus (HIV), severe acute respiratory syndrome    (SARS), poliovirus, human rhinovirus, adult T cell leukemia virus    (HTLV-I), hepatitis A, C, D, and E viruses, vaccinia virus, Japanese    encephalitis virus, dengue virus, human coronavirus, Ebola virus,    influenza virus, or sindbis virus, or (2) any one of the following    DNA viruses: a herpes simplex virus, human adenovirus, hepatitis B    virus, cytomegalovirus, EB virus, herpesvirus, human herpesvirus,    smallpox virus, polyoma virus, or human papilloma virus;-   [13] a method for screening for an antiviral agent, which comprises    the steps of: reacting a test compound with an SRPK, testing the    ability of the SRPK to phosphorylate an SR protein, and selecting a    compound that inhibits that ability;-   [14] the screening method of [13], which comprises the step of    testing the ability of an SRPK to phosphorylate an SR protein using,    as a substrate, an SR protein or a peptide with two or more    consecutive Arg-Ser (RS) or Ser-Arg (SR);-   [15] a method for producing antiviral agents, which comprises the    step of formulating a compound obtained by the method of [13] or    [14];-   [16] an aniline derivative represented by the following formula (I):

or a pharmaceutically acceptable salt or hydrate thereof;

-   wherein, R¹ represents a hydrogen atom, a C₁₋₆ alkyl group which may    have a substituent, a C₂₋₆ alkenyl group which may have a    substituent, a C₂₋₆ alkynyl group which may have a substituent, a    C₆₋₁₀ aryl group which may have a substituent, a halogen atom, a    nitro group, a cyano group, an azide group, a hydroxy group, a C₁₋₆    alkoxy group which may have a substituent, a C₁₋₆ alkylthio group    which may have a substituent, a C₁₋₆ alkylsulfonyl group which may    have a substituent, a carboxyl group, a formyl group, a C₁₋₆    alkoxycarbonyl group which may have a substituent, an acyl group, an    acylamino group, or a sulfamoyl group;-   R² represents a hydrogen atom, a C₁₋₆ alkyl group which may have a    substituent, or an aryl group which may have a substituent;-   R³ represents a C₁₋₆ alkyl group which may have a substituent, a    C₂₋₆ alkenyl group which may have a substituent, a C₆₋₁₀ aryl group    which may have a substituent, a nitrogen-containing heterocycle    which may have a substituent, or a condensed aromatic heterocycle    which may have a substituent;-   R⁴ represents a hydrogen atom or a halogen atom;-   Q represents —C(O)—, —C(S)—, —SO₂—, —C(S)NHC(O)—, —C(O)NHC(O)—, or    —C(O)NHC(S)—;-   W represents a hydrogen atom, a C₁₋₆ alkyl group which may have a    substituent, a C₆₋₁₀ aryl group which may have a substituent, a    halogen atom, a hydroxy group, a C₁₋₆ alkoxy group which may have a    substituent, a C₁₋₆ alkylthio group which may have a substituent, a    nitrogen-containing heterocycle which may have a substituent, a    condensed aromatic heterocycle which may have a substituent, or a    group represented by the following formula (II):

-   -   wherein, R⁵ and R⁶ are the same or different and each represents        a hydrogen atom, a C₁₋₆ alkyl group which may have a        substituent, a nitrogen-containing heterocycle which may have a        substituent, a condensed aromatic heterocycle which may have a        substituent, an acyl group, or an acylamino group;    -   the above R⁵ and R⁶ together with the adjacent nitrogen atom may        form a heterocycle which may have a substituent, and the        heterocycle may be a condensed aromatic heterocycle which may        have a substituent;    -   the above R⁵ and R⁶ may be a cycloalkylidene amino group which        may have a substituent, or an aromatic condensed cycloalkylidene        group which may have a substituent;

-   [17] the aniline derivative of [16], or a pharmaceutically    acceptable salt or hydrate thereof, wherein the above R¹ is a    hydrogen atom, a C₁₋₆ alkyl group which may have a substituent, or a    halogen atom;

-   [18] the aniline derivative of [16] or [17], or a pharmaceutically    acceptable salt or hydrate thereof, wherein the above R² is a    hydrogen atom or a C₁₋₆ alkyl group;

-   [19] the aniline derivative of any one of [16] to [18], or a    pharmaceutically acceptable salt or hydrate thereof, wherein the    above R³ is a C₆₋₁₀ aryl group which may have a substituent, or a    nitrogen-containing 5- to 10-membered heteroaryl group which may    have a substituent;

-   [20] the aniline derivative of any one of [16] to [19], or a    pharmaceutically acceptable salt or hydrate thereof, wherein the    above R⁴ is a hydrogen atom;

-   [21] the aniline derivative of any one of [16] to [20], or a    pharmaceutically acceptable salt or hydrate thereof, wherein the    above W represents a hydrogen atom, a halogen atom, or a group    represented by the following formula (II):

wherein, R⁵ and R⁶ are the same or different and each represent a C₁₋₆alkyl group which may have a substituent; or

-   the above R⁵ and R⁶ together with the adjacent nitrogen atom may    form a heterocyclic group which may have a substituent, and the    heterocyclic group may be a condensed aromatic heterocyclic group    which may have a substituent;-   [22] an SRPK inhibitor comprising as an active ingredient any one of    the aniline derivatives of [16] to [21], or a pharmaceutically    acceptable salt or hydrate thereof; and-   [23] an antiviral agent comprising as an active ingredient any one    of the aniline derivatives of [16] to [21], or a pharmaceutically    acceptable salt or hydrate thereof.

Inventions that comprise one or more combinations of inventions setforth in claims that cite an identical claim are intended to include theinventions of those claims

Hereinafter, the terms, symbols, and such used herein are defined, andthe present invention will be explained in more detail.

Herein, “C₁₋₆ alkyl group” refers to a linear or branched alkyl groupcomprising one to six carbon atoms, which is a monovalent group derivedby removing an arbitrary hydrogen atom from an aliphatic hydrocarbonconsisting of one to six carbons. Specifically, the C₁₋₆ alkyl groupincludes, for example, a methyl group, an ethyl group, a 1-propyl group,a 2-propyl group, a 2-methyl-1-propyl group, a 2-methyl-2-propyl group,a 1-butyl group, a 2-butyl group, a 1-pentyl group, a 2-pentyl group, a3-pentyl group, a 2-methyl-1-butyl group, a 3-methyl-1-butyl group, a2-methyl-2-butyl group, a 3-methyl-2-butyl group, a2,2-dimethyl-1-propyl group, a 1-hexyl group, a 2-hexyl group, a 3-hexylgroup, a 2-methyl-1-pentyl group, a 3-methyl-1-pentyl group, a4-methyl-1-pentyl group, a 2-methyl-2-pentyl group, a 3-methyl-2-pentylgroup, a 4-methyl-2-pentyl group, a 2-methyl-3-pentyl group, a3-methyl-3-pentyl group, a 2,3-dimethyl-1-butyl group, a3,3-dimethyl-1-butyl group, a 2,2-dimethyl-1-butyl group, a2-ethyl-1-butyl group, a 3,3-dimethyl-2-butyl group, and a2,3-dimethyl-2-butyl group.

Herein, “C₂₋₆ alkenyl group” refers to a linear or branched alkenylgroup comprising two to six carbons. Specifically, the C₂₋₆ alkenylgroup includes, for example, a vinyl group, an allyl group, a 1-propenylgroup, a 2-propenyl group, a 1-butenyl group, a 2-butenyl group, a3-butenyl group, a pentenyl group, and a hexenyl group.

Herein, “C₁₋₆ alkynyl group” refers to a linear or branched alkynylgroup comprising two to six carbons. Specifically, the C₂₋₆ alkynylgroup includes, for example, an ethynyl group, a 1-propynyl group, a2-propynyl group, a butynyl group, a pentynyl group, and a hexynylgroup.

Herein, “C₁₋₆ alkoxy group” refers to an oxy group to which theabove-defined “C₁₋₆ alkyl group” is linked. Specifically, the C₁₋₆alkoxy group includes, for example, a methoxy group, an ethoxy group, a1-propyloxy group, a 2-propyloxy group, a 2-methyl-1-propyloxy group, a2-methyl-2-propyloxy group, a 1-butyloxy group, a 2-butyloxy group, a1-pentyloxy group, a 2-pentyloxy group, a 3-pentyloxy group, a2-methyl-1-butyloxy group, a 3-methyl-1-butyloxy group, a2-methyl-2-butyloxy group, a 3-methyl-2-butyloxy group, a2,2-dimethyl-1-propyloxy group, a 1-hexyloxy group, a 2-hexyloxy group,a 3-hexyloxy group, a 2-methyl-1-pentyloxy group, a 3-methyl-1-pentyloxygroup, a 4-methyl-1-pentyloxy group, a 2-methyl-2-pentyloxy group, a3-methyl-2-pentyloxy group, a 4-methyl-2-pentyloxy group, a2-methyl-3-pentyloxy group, a 3-methyl-3-pentyloxy group, a2,3-dimethyl-1-butyloxy group, a 3,3-dimethyl-1-butyloxy group, a2,2-dimethyl-1-butyloxy group, a 2-ethyl-1-butyloxy group, a3,3-dimethyl-2-butyloxy group, and a 2,3-dimethyl-2-butyloxy group.

Herein, “C₁₋₆ alkylthio group” refers to a thio group to which theabove-defined “C₁₋₆ alkyl group” is linked. Specifically, the “C₁₋₆alkylthio group” includes, for example, a methylthio group, an ethylthiogroup, a 1-propylthio group, a 2-propylthio group, a butylthio group,and a pentylthio group.

Herein, “C₁₋₆ alkoxycarbonyl group” refers to a carbonyl group to whichthe above-defined “C₁₋₆ alkoxy group” is linked. Specifically, the C₁₋₆alkoxycarbonyl group includes, for example, a methoxy carbonyl group, anethoxy carbonyl group, a 1-propyloxycarbonyl group, and a2-propyloxycarbonyl group.

Herein, “C₁₋₆ alkylsulfonyl group” refers to a sulfonyl group to whichthe above-defined “C₁₋₆ alkyl group” is linked. Specifically, the C₁₋₆alkylsulfonyl group includes, for example, a methylsulfonyl group, anethylsulfonyl group, a 1-propylsulfonyl group, and a 2-propylsulfonylgroup.

Herein, “halogen atom” refers to a fluorine atom, a chlorine atom, abromine atom, and an iodine atom.

Herein, “C₆₋₁₀ aryl group” refers to an aromatic cyclic hydrocarbongroup comprising six to ten carbon atoms. Specifically, the C₆₋₁₀ arylgroup includes, for example, a phenyl group, a 1-naphthyl group, and a2-naphthyl group.

Herein, “heterocycle” refers to an aromatic or non-aromatic ring thatmay comprise double bonds within the ring, wherein one or two of theatoms constituting the ring are heteroatoms.

Herein, “nitrogen-containing heterocycle” refers to an aromatic ornon-aromatic ring that may comprise double bonds within the ring,wherein one or two of the atoms constituting the ring are nitrogenatoms.

Herein, “heteroatom” refers to a sulfur atom, an oxygen atom, or anitrogen atom.

Herein, “nitrogen-containing 5- to 10-membered heteroaryl ring” refersto an aromatic ring in which five to ten atoms constitute the ring,wherein at least one of the atoms constituting the ring is a nitrogenatom, and one or more heteroatoms other than nitrogen atoms may furtherbe comprised.

Specifically, the nitrogen-containing 5- to 10-membered heteroaryl ringincludes, for example, a pyridine ring, a pyrrole ring, an oxazole ring,an isoxazole ring, a thiazole ring, an isothiazole ring, an indole ring,an isoindole ring, an imidazole ring, a triazole ring, a pyrazole ring,a pyridazine ring, a pyrimidine ring, a pyrazine ring, a quinoline ring,an isoquinoline ring, and a benzimidazole ring.

The “5- to 10-membered heteroaryl ring” preferably includes a pyridinering, a pyrrole ring, and an imidazole ring, and more preferablyincludes a pyridine ring.

Herein, “nitrogen-containing 5- and 10-membered heteroaryl group” refersto a mono- or divalent group derived by removing one or two arbitraryhydrogen atoms from the above-defined “5- and 10-membered heteroarylring”. Specifically, the nitrogen-containing 5- and 10-memberedheteroaryl group includes, for example, a pyridyl group, a pyrrolylgroup, an oxazolyl group, an isoxazolyl group, a thiazolyl group, anisothiazolyl group, an indolyl group, an isoindolyl group, an imidazolylgroup, a triazolyl group, a pyrazolyl group, a pyridazinyl group, apyrimidinyl group, a pyrazinyl group, a quinolyl group, an isoquinolylgroup, and a benzimidazolyl group.

Herein, “4- to 8-membered heterocyclic ring” refers to a non-aromaticring that meets the following definition:

-   1. four to eight atoms constitute the ring;-   2. one or two of the atoms constituting the ring are heteroatoms;-   3. one or two double bonds may be comprised in the ring;-   4. one to three carbonyl groups may be comprised in the ring; and-   5. the group is monocyclic.

The 4- to 8-membered heterocyclic ring is preferably anitrogen-containing 4- to 8-membered heterocyclic ring that comprisesnitrogen atoms as heteroatoms.

Specifically, the 4- to 8-membered heterocyclic ring includes, forexample, an azetidine ring, a pyrrolidine ring, a piperidine ring, anazepane ring, an azocine ring, a tetrahydropyran ring, a morpholinering, a thiomorpholine ring, a piperazine ring, a thiazolidine ring, adioxane ring, an imidazoline ring, and a thiazoline ring. The “4- to8-membered heterocyclic ring” preferably includes a pyrrolidine ring, apiperidine ring, a morpholine ring, and a piperazine ring.

Herein, “a 4- to 8-membered heterocyclic group” refers to a mono- ordivalent group derived by removing one or two arbitrary hydrogen atomsfrom the above-defined “4- to 8-membered heterocyclic ring”.Specifically, the 4- to 8-membered heterocyclic group includes, forexample, an azetidinyl group, a pyrrolidinyl group, a piperidinyl group,an azepanyl group, an azocanyl group, a tetrahydropyranyl group, amorpholinyl group, a thoimorpholinyl group, a piperazinyl group, athiazolidinyl group, a dioxanyl group, an imidazolyl group, and athiazolyl group.

Herein, “condensed aromatic heterocycle” refers to a ring structure inwhich the heterocyclic moiety is ortho-condensed with an aromatic ring,such as a benzene ring. The heterocyclic moiety is an above-definedheterocycle.

Herein, “condensed aromatic heterocyclic group” refers to a ringstructure in which the heterocyclic moiety is ortho-condensed with anaromatic ring, such as benzene ring. The heterocyclic moiety is anabove-defined heterocyclic group.

The condensed aromatic heterocyclic group includes, for example, anindolinyl group, an isoindolinyl group, and a1,2,3,4-tetrahydroquinoline.

Herein, “halogenated C₁₋₆ alkyl group” refers to a group in which atleast one arbitrary hydrogen atom in the above-defined “C₁₋₆ alkylgroup” is replaced with an above-defined “halogen atom”. The halogenatedC₁₋₆ alkyl group includes, for example, a trifluoromethyl group, adifluoromethyl group, and a monofluoromethyl group.

Herein, the phrase “may have substituents” means that a certain compoundmay have an arbitrary combination of one or more substituents atsubstitutable positions. Specifically, the substituents include, forexample, groups selected from the following Substituent Group A:

[Substituent group A]

a halogen atom, a hydroxyl group, a mercapto group, a nitro group, acyano group, a formyl group, a carboxyl group, a trifluoromethyl group,a trifluoromethoxy group, an amino group, an oxo group, an imino group,a C₁₋₆ alkyl group, a C₁₋₆ alkoxy group.

Herein, “salt” is not particularly limited, so long as it is apharmaceutical acceptable salt which is formed with a compound accordingto the present invention. Such salts include, for example, inorganicacid salts, organic salts, inorganic base salts, organic base salts, andacidic or basic amino acid salts.

Examples of preferable inorganic acid salts include: hydrochloride,hydrobromate, sulfate, nitrate, and phosphate. Examples of preferableorganic salts include: acetate, succinate, fumarate, maleate, tartrate,citrate, lactate, stearate, benzoate, methanesulfonate, and p-toluenesulfonate.

Examples of preferable inorganic base salts include: alkali metal salts,such as sodium salts and potassium salts; alkali earth metal salts, suchas calcium salts and magnesium salts; aluminium salts; and ammoniumsalts. Examples of preferable organic base salts include: diethylaminesalts, diethanol amine salts, meglumine salts, andN,N′-dibenzylethylenediamine salts.

Examples of preferable acidic amino acid salts include: aspartate andglutamate. Examples of preferable basic amino acid salts include:arginine salts, lysine salts, and ornithine salts.

When left in air, the compounds of the present invention sometimesabsorb moisture, and are sometimes attached to absorbed water orconverted to hydrates. Such hydrates are also included in the presentinvention.

Furthermore, compounds of the present invention are sometimes convertedinto solvates, absorbing some other solvents. Such salts are alsoincluded in the present invention.

Herein, “gene” refers to DNAs or RNAs encoding transcriptional units insense or antisense orientation. Transcriptional units refer to sequencesthat are continuously transcribed. Herein, a nucleic acid (DNA or RNA)encoding a protein is also referred to as a “gene for that protein”.

Herein, the term “or” is used non-exclusively. For example, the phrase“A, B, or C” means that at least any one element of A, B, and C iscomprised, and therefore the phrase also comprises things that comprisetwo or more of, or all three of A, B, and C, and things that compriseother elements.

Herein, the compounds shown in Tables 1 to 3 are sometimes referred bycompound number. These compounds are sometimes shown as “GIF-” , citinga compound number.

Effects of the Invention

The present invention revealed that SRPIN-1 (SR protein phosphorylationinhibitor 1) and analogs thereof had the activity of inhibiting SRPKs,which are kinases. SR proteins phosphorylated by SRPKs were found toexist stably in cells; however, SR protein phosphorylation is inhibitedwhen SRPK enzyme activity is inhibited by SRPIN-1 or analogs and suchthereof, leading to degradation of SR proteins via theubiquitin-proteasome pathway. Then, the inventors inhibited SRPKs byadding SRPIN-1 or analogs thereof, and thus discovered that thesecompounds had the antiviral activity of inhibiting viral replication inHIV infection experiments.

The present invention is also beneficial in that it provides antiviralagents that control the activity of SR proteins, and by the samemechanism, are effective against a broad range of viruses.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A: Phosphorylation of SR protein in HIV-infected cells. The pNL4-3genome was introduced into Flp-In-293 cells. SR protein phosphorylationin these Flp-In-293 cells was evaluated by Western analysis using mouseanti-phosphorylated SR protein monoclonal antibody (Mab104), mouseanti-SC35 antibody, and mouse anti-SF2 antibody.

FIG. 1B: Degradation of SR protein. Plasmids for the SRp75, SRp55, andSRp40 genes were fused with HA tag and introduced into Flp-In-293 cells.MG132 (474790; purchased from CALBIOCHEM) was added to the cells at afinal concentration of 10 μM. The cells were lysed and heat-denatured.The resulting protein sample was separated by SDS-PAGE, followed byWestern analysis using a rabbit anti-HA antibody as the primary antibodyand a donkey anti-rabbit IgG antibody as the secondary antibody.

FIG. 2A: Phosphorylation of SR protein in cells stably expressing SRPK2.Mouse SRPK2 gene was introduced into Flp-In-293 cells to prepare cellsstably expressing SRPK2 (SRPK2-2). pNL4-3 was introduced into theseSRPK2-2 cells and cells of the parent cell line Flp-In-293 (mock). Afterfour days, the kinetics of endogenous SR protein during HIV infectionwas evaluated by Western analysis in the same way as in FIG. 1A.

FIG. 2B: Existence of SR protein in cells stably expressing SRPK2. TheHIVpNL4-3 genome and plasmids for SRp75, SRp55, and SRp40 genes fusedwith HA tag were introduced into mock and SRPK2-2 cells, prepared as inFIG. 2A. The samples were harvested after 36 hours and analyzed byWestern blotting.

FIG. 2C: Measuring the quantity of produced HIV. The culturesupernatants obtained as described in FIG. 2A were collected and theamount of produced HIV was determined.

FIG. 3A: Evaluation of SR protein contributing to in-vivo HIVproduction. Mock plasmid, and the SC35, SF2, SRp40, SRp55, and SRp75expression plasmids were each introduced into Flp-In293 cells. After 36hours, culture supernatants were collected and the amount of HIVp24 weredetermined using the Lumipulse ELISA system.

FIG. 3B: Evaluation of the effect of hnRNPA1 on in-vivo HIV production.Gene transfer into Flp-In293 cells was carried out using a fixed amount(500 ng) of an SRp40 or SRp75 expression plasmid as well as an hnRNPA1expression plasmid, the amount of which was step increased. After 36hours, culture supernatants were collected and the amount of HIVp24 wasdetermined using the Lumipulse ELISA system.

FIG. 4A: Search for SRPK inhibitors to inhibit the phosphorylation ofintracellular SR protein. Structural formula of SRPIN-1 (SRPkInhibitor-1).

FIG. 4B: Evaluation of inhibition of the phosphorylation activity ofSRPK1 by SRPIN-1. An RS peptide corresponding to RS domain of SF2 wasdissolved in 10 mM Tris-HCl to a concentration of 1 mg/ml (pH 7.5). Thepeptide was incubated for ten minutes with 1 μg of SRPK1 protein in areaction buffer (250 μM MgCl₂, 0.25 mM ATP, 1 mCi of [γ-³²P]ATP, andSRPIN-1 (final concentration: 0.1, 0.3, 1.0, 3.0, or 10.0 μM)) in awater bath at 30° C. The reaction mixture was dropped onto a P81phosphocellulose membrane (P81; Whatman), and then the membrane waswashed with a 5% phosphoric acid solution. After washing, theradioactivity of ³²P on the P81 membrane was determined in a liquidscintillation counter.

FIG. 4C: Evaluation of in-vivo inhibition of SR protein phosphorylationusing SRPIN-1, and evaluation of the induction of the accompanying SRprotein degradation. HA-SRp75 plasmid was introduced into Flp-In293cells. After 36 hours, MG132 (final concentration: 10 μM) and SRPIN-1(10, 20, or 50 μM) were added to the cells. The cells were incubated for15 hours and then lysed. After SDS-PAGE, the samples were analyzed byWestern blotting using anti-HA antibody. Western analysis was alsocarried out using an antibody against beta actin as a control proteinamount.

FIG. 4D: Evaluation of the inhibition of HIV infection upon addition ofSRPIN-1. HIV virion was prepared using 293 T cells and then added toMT-4 cells along with SRPIN-1 (final concentration: 0.5, 10, or 20 μM).After two hours of incubation at 37° C. under 5% CO₂, the cells werecentrifuged to change the culture medium. The culture supernatant wascollected after 48 hours, and the amount of HIVp24 was determined by theLumipulse ELISA system.

FIG. 5A: Evaluation of the SRPK-inhibiting activities of SRPIN-1 andanalogs thereof. The inhibitory effects of SRPIN-1 (Compound No. 340)and analogs thereof (Compound Nos. 341 to 349, and 608 to 626) on thephosphorylation activities of SRPK1 and SRPK2 were determined.

FIG. 5B: The effect of SRPIN-1 and analogs thereof in inhibiting HIVreplication. This figure shows the results of assaying the effect ofSRPIN-1 and analogs thereof in inhibiting HIV replication in MT-4 cells.

FIG. 5C: The effect of SRPIN-1 and analogs thereof in inhibiting HIVreplication. Like FIG. 5B, this figure shows the results of assaying theeffect of SRPIN-1 and analogs thereof in inhibiting HIV replication inJurkat cells.

FIG. 6A: Antiviral activity of SRPIN-1 against sindbis virus. Thisfigure shows phase contrast microscopic images of cells infected withsindbis virus. Marked cell damage caused by the propagation of sindbisvirus was found in cells to which SRPIN-1 was not administered, whilecell damage was dramatically inhibited by administering SRPIN-1.

FIG. 6B: Antiviral activity of SRPIN-1 against sindbis virus. Thisfigure shows the results of a plaque assay for sindbis virus-infectedcells. SRPIN-1 significantly inhibited the propagation of Sindbis virusin a concentration-dependent manner when its concentration was 5 μM orhigher.

FIG. 7: Antiviral activity of SRPIN-1 and analogs thereof againstcytomegalovirus. This figure shows phase contrast microscopic images ofcytomegalovirus-infected cells. Morphological alterations characteristicof cytomegalovirus infection and cell death were frequently found incontrol group cells (1 and 2 in this figure). In contrast, the abnormalmorphological alterations and cell death caused by cytomegalovirusinfection were inhibited in cells to which 20 μl of SRPIN-1 or an analogthereof (Compound No. 349) was added (3 and 5 in this figure).

FIG. 8: Antiviral activities of SRPIN-1 and analogs thereof against SARSvirus. This figure shows the results of plaque assays for SARSvirus-infected cells. The number of plaques where SARS virus infectionresulted in cell death was determined to evaluate the antiviral activityof SRPIN-1 and analogs thereof (plaque assay). As a result, 40 μMSRPIN-1 and analog compounds thereof (Compound No. 349) significantlyinhibited SARS virus propagation, as shown in FIG. 8A. In addition, asshown in FIG. 8B, SRPIN-1 was found to inhibit SARS virus propagation ina concentration-dependent manner within a concentration range of 1 to 40μM.

BEST MODE FOR CARRYING OUT THE INVENTION

The present inventors investigated whether antiviral agents against abroad range of viruses could be provided by broadly applying thephenomenon in which HIV replication can be inhibited by inhibiting SRPKenzymes, which phosphorylate SR proteins.

I. SR Proteins

Activity Reduction: Degradation and Stabilization

(1) The present inventors investigated the relationship between theinfection of cells with HIV virus, and the phosphorylation state of SRprotein and SR protein presence in cells. Specifically, 293 cells wereinfected with type NL4-3 HIV virus, and then the total amounts of SRprotein in the cells and phosphorylated protein in the cells weremeasured using antibodies against SR protein and against phosphorylatedSR protein.

Furthermore, the present inventors also investigated the relationshipbetween HIV infection of cells forced to express SRPK, whichphosphorylates SR protein, and the phosphorylation state of SR proteinand existence of SR protein in these same cells. Specifically, in asimilar way to the above, 293 cells forced to express SRPK-2 wereinfected with type NL4-3 HIV virus. The total amounts of SR protein inthe cells and phosphorylated protein in the cells were then measuredusing antibodies against SR protein and against phosphorylated SRprotein.

The results described above show that phosphorylated SR protein isstabilized in cells, but SR protein can be degraded whendephosphorylated.

Further, to confirm the above conclusion, the present inventorsexpressed SR-HA fusion protein in 293 cells, and measured the signalintensity of fusion SR-HA when reacted with an anti-HA antibody in theabsence or presence of MG132, a ubiquitin proteasome inhibitor. Theinventors found that MG132 inhibited protein degradation, and thus theSR protein was degraded via the ubiquitin-proteasome pathway.

Specifically, the present inventors speculated that hosts degrade SRprotein as a defense mechanism in response to viral infection. However,when SR protein kinase was forcedly expressed, the SR protein was notdegraded, but instead stabilized by the phosphorylation. Thisstabilization was found to override the defense mechanism, contributingto enhanced viral production.

Specifically, the present inventors found that SR protein was degradedby ubiquitin proteasome when dephosphorylated. Since SR protein isessential for gene transcription, dephosphorylating the SR protein caninhibit viral propagation.

(2) The present inventors next investigated the inhibition of SR proteinkinase. SRPK1/2, Clk/Sty family kinase, PRP4, DNA topoisomerase I, andothers are thought to be candidate kinase responsible forphosphorylating SR proteins, but much is unclear regarding theirfunctional differences in terms of splicing. Thus, the present inventorsinvestigated viral production in virus-infected cells when SRPK wasinhibited using SRPIN-1, an SRPK inhibitor. Inhibition of SRPK bySRPIN-1 was found to induce active degradation of SR protein.

(3) Cells were again infected with HIV, and at the same time forced toexpress hnRNPA1, which is known to antagonize in vitro SR protein, whichpromotes splicing. As a result, the present inventors discovered for thefirst time that hnRNPA1 inhibited in vivo HIV production in adose-dependent manner, while SRp40 and SRp75 further promoted HIVproduction.

As described above, the dephosphorylation of SR protein is a biologicaldefense reaction against viruses (in the human body). It had beenpreviously confirmed that SR protein was dephosphorylated in animalcells after infection with adenovirus or vaccinia virus (Nature Vol.393, pp. 185-187, EMBO Rep Vol. 3, pp. 1088-1093). Thus, as describedabove, it is thought that upon dephosphorylation the SR protein israpidly degraded, and thus becomes unavailable for viral geneexpression. As a result, the viruses cannot propagate.

The present inventors confirmed that inhibition of SR protein activityby SRPK inhibitors resulted in the inhibition of propagation of not onlyHIV but also sindbis virus, cytomegalovirus, and SARS coronavirus, whichare viruses different from HIV. Thus, it can be concluded that theantiviral action generated by controlling the activity of SR protein iseffective against a broad range of viruses.

II.

The present invention comprises antiviral agents that control theactivity of SR proteins, methods for inhibiting viral production, andmethods for treating viral diseases. The present invention comprisesantiviral agents whose active ingredients comprise agents that controlSR protein activity. Control of SR protein activity also comprisescontrol of expression and stabilization. For example, SR proteinactivity can be reduced by inhibiting the transcription or translationof SR proteins, or reducing the stability of SR proteins or mRNAsencoding SR proteins. Preferably, when controlling the activity of SRproteins as per the present invention, the SR protein activityinhibitors directly or indirectly reduce the activity or expressionlevel of SR proteins. To reduce the activity or expression level of anSR protein, for example, in addition to using the SR protein as a directtarget, it is also preferable to inhibit phosphorylation of the SRprotein by SRPKs and/or enhance dephosphorylation. Dephosphorylation ofSR proteins is promoted, for example, by activating protein phosphatase2A (also referred to as phosphatase 2A). Thus, viral propagation can beinhibited by using a compound that increases the expression and/oractivity of protein phosphatase 2A. Phosphorylation of SR proteins canalso be inhibited by inhibiting the expression and/or activity of SRPKs.Thus, SRPK inhibitors are preferable antiviral agents of the presentinvention.

[SR Proteins as Targets for Control]

The SR proteins whose activity is to be reduced or inhibited accordingto the present invention may be arbitrary SR proteins, and specificallyinclude X16/SRp20, SF2/ASF/SRp30a, SC35/PR264/SRp30b, SRp30c, 9G8,HRS/SRp40, SRp46, SRp55, SRp75, and p54. Preferable SR proteins areSF2/ASF/SRp30a, SC35/PR264/SRp30b, SRp30c, HRS/SRp40, SRp46, and SRp75,and particularly preferable SR proteins are SRp40 and SRp75.Hereinafter, SR protein refers to SF2/ASF/SRp30a, SC35/PR264/SRp30b,SRp30c, HRS/SRp40, SRp46, or SRp75.

The gene sequence encoding X16/SRp20 is set forth, for example, innucleotides 1-492 of accession number L10838. The amino acid sequence ofX16/SRp20 is set forth in accession numbers NP_(—)003008 and AAA36648(Zahler, A. et al., 1992, SR proteins: a conserved family of pre-mRNAsplicing factors, Genes Dev. 6:837-847). The gene sequence encodingSF2/ASF/SRp30a is set forth, for example, in nucleotides 91-834 ofaccession number NM_(—)006924. The amino acid sequence of SF2/ASF/SRp30ais set forth in accession numbers NP_(—)008855 and Q07955 (Ge, H. etal., Cell 66, 373-382 (1991)). The gene sequence encodingSC35/PR264/SRp30b is set forth, for example, in nucleotides 156-818 ofaccession number M90104. The amino acid sequence of SC35/PR264/SRp30b isset forth in accession numbers AAA60306 and Q01130 (Fu, X. D. andManiatis, T. Science 256, 535-538 (1992)). The gene sequence encodingSRp30c is set forth, for example, in nucleotides 53-715 and nucleotides147-809 of accession numbers U30825 and NM_(—)003769, respectively. Theamino acid sequence of SRp30c is set forth in accession numbersAAA93069, Q13242, and NP_(—)003760 (Screaton, G. R. et al., EMBO J. 14,4336-4349 (1995)). The gene sequence encoding 9G8 is set forth, forexample, in nucleotides 54-464 of accession number NM_(—)006276. Theamino acid sequence of 9G8 is set forth in accession numbersNP_(—)006267, Q16629, and such (Lejeune, F. et al., J. Biol. Chem. 276,7850-7858 (2001); Popielarz, M. et al., J. Biol. Chem. 270, 17830-17835(1995); Cavaloc, Y. et al., EMBO J. 13, 2639-2649 (1994)). The genesequence encoding HRS/SRp40 is set forth, for example, in accessionnumber AF020307 (join(2406-2531, 2864-2925, 3049-3147, 3433-3503,4740-4812, 5269-5382, 5472-5492)), and the amino acid sequence is setforth in accession numbers AAC39543 and Q13243, and other (Du, K. andTaub, R., Gene 204 (1-2), 243-249 (1997); Screaton, G. R. et al., EMBOJ. 14, 4336-4349 (1995)). The gene sequence encoding SRp46 is set forth,for example, in nucleotides 1-816 of accession number AF031166. Theamino acid sequence of SRp46 is set forth in accession number AAK54351and others (Soret, J. et al., Mol. Cell. Biol. 18, 4924-4934 (1998)).The gene sequence encoding SRp55 is set forth, for example, innucleotides 106-1137 of accession number U30883. The amino acid sequenceof SRp55 is set forth in accession numbers AAA93073 and Q13247, andothers (Screaton, G. R. et al., EMBO J. 14, 4336-4349 (1995); Zahler, A.M. et al., Genes Dev. 6, 837-847 (1992); Barnard, D. C. and Patton, J.G., Mol. Cell. Biol. 20, 3049-3057 (2000)). The gene sequence encodingSRp75 is set forth, for example, in nucleotides 98-1579 and nucleotides98-1579 of accession numbers BC002781 and NM_(—)005626, respectively.The amino acid sequence of SRp75 is set forth in accession numbersAAH02781, NP_(—)005617 and Q08170, and others (Zahler, A. M. et al.,Mol. Cell. Biol. 13, 4023-4028 (1993)). The gene sequence encoding p54is set forth, for example, in nucleotides 84-1535 of accession numberM74002. The amino acid sequence of p54 is set forth in accession numbersAAA35554 and Q05519, and others (Chaudhary, N. et al., Proc. Natl. Acad.Sci. U.S.A. 88, 8189-8193 (1991)).

[Target Viruses]

The antiviral agents of the present invention particularly preferablyinhibit HIV propagation, but are not limited to human immunodeficiencyvirus (HIV) and also have a similar effect on other viruses, includingRNA viruses, such as severe acute respiratory syndrome (SARS),polioviruses, human rhinoviruses, adult T cell leukemia viruses(HTLV-I), hepatitis A, C, D, and E viruses (excluding hepatitis Bvirus), vaccinia viruses, Japanese encephalitis viruses, dengue viruses,human coronaviruses, Ebola viruses, influenza viruses, and sindbisviruses. Human coronaviruses include SARS coronaviruses (also referredto as a SARS-associated coronavirus or SARS virus).

Since SR protein dephosphorylation has been reported as a host defensemechanism upon infection of herpes simplex viruses and humanadenoviruses, which are DNA viruses, SRPIN-1 affects herpes simplexviruses and human adenoviruses, and also has a similar effect onhepatitis B viruses, cytomegaloviruses, EB viruses, herpesviruses, humanherpes viruses, smallpox viruses, polyoma viruses, and human papillomaviruses.

Particularly preferable target viruses of the present invention includeviruses of the retrovirus family (Retroviridae; including viruses of thegenus lentivirus), togavirus family (Togaviridae; including viruses ofthe genus alphavirus), herpesvirus family (Herpesviridae; includingcytomegalovirus), and coronavirus family (Coronaviridae; includingviruses of the genus coronavirus).

[Antiviral Agents]

The present invention includes: (1) antiviral agents that act byreducing or inhibiting SR protein activity, more specifically, antiviralagents that act by enhancing the dephosphorylation of SR proteins, and(ii) antiviral agents that act by inhibiting proteins that phosphorylateSR proteins.

The present invention also includes: (2) antiviral agents that act byinhibiting SR protein expression, and (3) antiviral agents that act byactivating the function of proteins that antagonize SR proteins.

In particular, the present invention relates to antiviral agentscomprising compounds that inhibit the activity and/or expression ofSRPK. The phosphorylation that contributes to the stabilization of SRprotein is inhibited by inhibiting the activity and/or expression ofSRPK. As a result, degradation of SR protein is promoted, and SR proteinactivity is reduced. Thus, SRPK (SRPK1 and/or SRPK2) is a particularlypreferable inhibition target of the present invention.

[Methods for Inhibiting Viral Production]

The present invention also includes: (1) methods for inhibiting viralproduction by reducing or inhibiting SR protein activity, morespecifically, the present invention includes (i) methods for inhibitingvirus production by enhancing the dephosphorylation of SR protein, and(ii) methods for inhibiting virus production by inhibiting proteins thatphosphorylate SR protein. In particular, the present invention relatesto methods for inhibiting viral production, which comprise the step ofinhibiting the activity and/or expression of SRPK. When SRPK isinhibited, the phosphorylation of SR protein is inhibited, and the SRprotein level is reduced, which thus reduces SR protein activity.

The present invention also includes: (2) methods for inhibiting viralproduction by inhibiting SR protein expression, and (3) methods forinhibiting viral production by activating the function of proteins thatantagonize SR protein.

Specifically, the present invention also includes the followinginventions:

-   [M1] Method of inhibiting propagation of a virus, which comprises    the step of reducing an activity or expression level of an SR    protein;-   [M2] the method of [M1], in which the SR protein is SF2/ASF/SRp30a,    SC35/PR264/SRp30b, SRp30c, HRS/SRp40, SRp46, or SRp75;-   [M3] the method of [M1] or [M2], in which the step of reducing an    activity or expression level of an SR protein is the step of    inhibiting the phosphorylation of an SR protein or enhances its    dephosphorylation;-   [M4] the method of [M3], in which the step of inhibiting the    phosphorylation of an SR protein or enhances its dephosphorylation    is the step of increasing an activity of protein phosphatase 2A;-   [M5] the method of [M4], in which the step of increasing an activity    of protein phosphatase 2A is the step of introducing an expression    vector for one or more genes selected from the group consisting of:    an HIV tat gene, adenovirus E4-ORF4 gene, and vaccinia virus VH1    gene;-   [M6] the method of [M3], in which the step of inhibiting the    phosphorylation of an SR protein or enhances its dephosphorylation    is the step of inhibiting an expression or activity of a SRPK;-   [M7] the method of [M6], in which the SRPK is SRPK1 or SRPK2;-   [M8] the method of [M6] or [M7], in which the step of inhibiting an    expression or activity of a SRPK is the step of administering an    aniline derivative represented by the following formula:

or a pharmaceutically acceptable salt or hydrate thereof;

-   wherein, R¹, R², R³, R⁴, Q, and W are defined in [16] herein above;-   [M9] the method of [M8], in which the above R¹ is a hydrogen atom, a    C₁₋₆ alkyl group which may have a substituent, or a halogen atom;-   [M10] the method of [M8] or [M9], in which the above R² is a    hydrogen atom or a C₁₋₆ alkyl group;-   [M11] the method of any one of [M8] to [M10], in which the above R³    is a C₆₋₁₀ aryl group which may have a substituent, or a    nitrogen-containing 5- to 10-membered heteroaryl group which may    have a substituent;-   [M12] the method of any one of [M8] to [M11], in which the above R⁴    is a hydrogen atom;-   [M13] the method of any one of [M8] to [M12], in which the above W    is a hydrogen atom, a halogen atom, or a group represented by the    following formula (II):

wherein, R⁵ and R⁶ are defined above;

-   [M14] the method of [M8], in which the aniline derivative of [M8] is    selected from the group consisting of compounds with Compound Nos:    340, 348, 613, 616, 618, 622, and 624 described herein;-   [M15] the method of [M6], in which the step of inhibiting an    expression or activity of a SRPK is the step of introducing a SRPK    miRNA, siRNA or morpholino oligo, or introducing an miRNA or siRNA    expression vector;-   [M16] the method of [M1] or [M2], in which the step of reducing an    activity or expression level of an SR protein is the step of    administering a substance having an activity of antagonizing an SR    protein;-   [M17] the method of [M16], in which the substance having an activity    of antagonizing an SR protein is an hnRNP A1 expression vector;-   [M18] the method of any one of [Ml] to [M17], in which the virus is:    any one of (1) an RNA virus: a human immunodeficiency virus (HIV),    severe acute respiratory syndrome (SARS), poliovirus, human    rhinovirus, adult T cell leukemia virus (HTLV-I), hepatitis A, C, D,    and E virus, vaccinia virus, Japanese encephalitis virus, dengue    virus, human coronavirus, Ebola virus, influenza virus, and sindbis    virus, and (2) a DNA virus: a herpes simplex virus, human    adenovirus, hepatitis B virus, cytomegalovirus, EB virus,    herpesvirus, human herpesvirus, smallpox virus, polyoma virus, and    human papilloma virus.-   [M19] the method of inhibiting a SRPK, which comprises the step of    administering the aniline derivative of [M8], or a pharmaceutically    acceptable salt or hydrate thereof;-   [M20] the method of [M19], in which the SRPK is SRPK1 or SRPK2;-   [M21] the method of [M19] or [M20], in which the above R¹ is a    hydrogen atom, a C₁₋₆ alkyl group which may have a substituent, or a    halogen atom;-   [M22] the method of any one of [M19] to [M21], in which the above R²    is a hydrogen atom or a C₁₋₆ alkyl group;-   [M23] the method of any one of [M19] to [M22], in which the above R³    is a C₆₋₁₀ aryl group which may have a substituent, or a    nitrogen-containing 5- to 10-membered heteroaryl group which may    have a substituent;-   [M24] the method of any one of [M19] to [M23], in which the above R⁴    is a hydrogen atom;-   [M25] the method of any one of [M19] to [M24], in which the above W    is a hydrogen atom, a halogen atom, or a group represented by the    following formula (II):

wherein, R⁵ and R⁶ are defined above; and

-   [M26] the method of [M19], in which the aniline derivative of [M8]    is a compound selected from the group consisting of compounds with    the Compound Nos: 340, 348, 613, 616, 618, 622, and 624 described    herein, or a pharmaceutically acceptable salt or hydrate thereof.

The present invention further relates to uses of the compounds thatreduce the expression and/or activity of SR protein for inhibiting viralpropagation and for producing antiviral agents (reagents and/orpharmaceuticals for inhibiting viral propagation). Specifically, thepresent invention also relates to the following inventions:

-   [U1] Use of a compound that reduces an activity or expression level    of an SR protein for inhibiting propagation of a virus or for    producing an antiviral agent;-   [U2] the use of [U1], in which the SR protein is SF2/ASF/SRp30a,    SC35/PR264/SRp30b, SRp30c, HRS/SRp40, SRp46, or SRp75;-   [U3] the use of [U1] or [U2], in which the compound that reduces an    activity or expression level of an SR protein is a compound that    inhibits the phosphorylation of an SR protein or enhances its    dephosphorylation;-   [U4] the use of [U3], in which the compound that inhibits the    phosphorylation of an SR protein or enhances its dephosphorylation    is a compound that increases an activity of protein phosphatase 2A;-   [U5] the use of [U4], in which the compound that increases an    activity of protein phosphatase 2A is an expression vector for one    or more genes selected from the group consisting of: an HIV tat    gene, adenovirus E4-ORF4 gene, and vaccinia virus VH1 gene;-   [U6] the use of [U3], in which the compound that inhibits the    phosphorylation of an SR protein or enhances its dephosphorylation    is a compound that inhibits an expression or activity of a SRPK;-   [U7] the use of [U6], in which the SRPK is SRPK1 or SRPK2;-   [U8] the use of [U6], in which the compound that inhibits an    expression or activity of a SRPK is an aniline derivative    represented by the following formula:

or a pharmaceutically acceptable salt or hydrate thereof;

-   wherein, R¹, R², R³, R⁴, Q, and W are defined in [16] herein above;-   [U9] the use of [U8], in which the above R¹ is a hydrogen atom, a    C₁₋₆ alkyl group which may have a substituent, or a halogen atom;-   [U10] the use of [U8] or [U9], in which the above R² is a hydrogen    atom or a C₁₋₆ alkyl group;-   [U11] the use of any one of [U8] to [U10], in which the above R³ is    a C₆₋₁₀ aryl group which may have a substituent, or a    nitrogen-containing 5- to 10-membered heteroaryl group which may    have a substituent;-   [U12] the use of any one of [U8] to [U11], in which the above R⁴ is    a hydrogen atom;-   [U13] the use of any one of [U8] to [U12], in which the above W is a    hydrogen atom, a halogen atom, or a group represented by the    following formula (II):

wherein, R⁵ and R⁶ are defined above;

-   [U14] the use of [U8], in which the aniline derivative of [U8] is    selected from the group consisting of compounds with Compound Nos:    340, 348, 613, 616, 618, 622, and 624 described herein;-   [U15] the use of [U6], in which the compound that inhibits an    expression or activity of a SRPK is a SRPK miRNA, siRNA or    morpholino oligo, or is an miRNA or siRNA expression vector;-   [U16] the use of [U1] or [U2], in which the compound that reduces an    activity or expression level of an SR protein is a substance having    an activity of antagonizing an SR protein;-   [U17] the use of [U16], in which the substance having an activity of    antagonizing an SR protein is an hnRNPA1 expression vector;-   [U18] the use of any one of [U1] to [U17], in which the virus is:    any one of (1) an RNA virus: a human immunodeficiency virus (HIV),    severe acute respiratory syndrome (SARS), poliovirus, human    rhinovirus, adult T cell leukemia virus (HTLV-I), hepatitis A, C, D,    and E virus, vaccinia virus, Japanese encephalitis virus, dengue    virus, human coronavirus, Ebola virus, influenza virus, and sindbis    virus, and (2) a DNA virus: a herpes simplex virus, human    adenovirus, hepatitis B virus, cytomegalovirus, EB virus,    herpesvirus, human herpesvirus, smallpox virus, polyoma virus, and    human papilloma virus.-   [U19] the use of the aniline derivative of [U8], or a    pharmaceutically acceptable salt or hydrate thereof for inhibiting a    SRPK or for producing a SRPK inhibitor;-   [U20] the use of [U19], in which the SRPK is SRPK1 or SRPK2;-   [U21] the use of [U19] or [U20], in which the above R¹ is a hydrogen    atom, a C₁₋₆ alkyl group which may have a substituent, or a halogen    atom;-   [U22] the use of any one of [U19] to [U21], in which the above R² is    a hydrogen atom or a C₁₋₆ alkyl group;-   [U23] the use of any one of [U19] to [U22], in which the above R³ is    a C₆₋₁₀ aryl group which may have a substituent, or a    nitrogen-containing 5- to 10-membered heteroaryl group which may    have a substituent;-   [U24] the use of any one of [U19] to [U23], in which the above R⁴ is    a hydrogen atom;-   [U25] the use of any one of [U19] to [U24], in which the above W is    a hydrogen atom, a halogen atom, or a group represented by the    following formula (II):

wherein, R⁵ and R⁶ are defined above; and

-   [U26] the use of [U19], in which the aniline derivative of [U8] is a    compound selected from the group consisting of compounds with the    Compound Nos: 340, 348, 613, 616, 618, 622, and 624 described    herein, or a pharmaceutically acceptable salt or hydrate thereof.    [Therapeutic Methods]

The present invention includes: (1) methods for treating or preventingviral diseases by reducing or inhibiting SR protein activity, morespecifically, (i) methods for treating or preventing viral diseases bydephosphorylating SR protein, and (ii) methods for treating viraldiseases by inhibiting proteins that phosphorylate SR protein. Inparticular, the present invention relates to methods for treating orpreventing viral diseases, which comprise the step of inhibiting theactivity and/or expression of SRPK. SRPK inhibition results ininhibition of SR protein phosphorylation, which reduces the SR proteinlevel and thus inhibits viral propagation.

The present invention also includes: (2) methods for treating orpreventing viral diseases by inhibiting the expression of SR protein,and (3) methods for treating or preventing viral diseases by activatingthe function of proteins that antagonize SR protein.

III.

The present invention also includes methods for screening for antiviralagents and uses of SRPK inhibitors.

[Methods for Screening for Antiviral Agents]

The present invention also includes: (1) methods for screening SRPKinhibitors using SR proteins or peptides with two or more consecutiveunits of RS or SR as SRPK substrates

[SRPK Inhibitors and Use Thereof]

The present invention also includes: (1) SRPK inhibitors comprisingSRPIN-1 or an analog thereof as an active ingredient, (2) viralpropagation inhibitors comprising SRPIN-1 or an analog thereof as anactive ingredient, and (3) antiviral therapeutic agents comprisingSRPIN-1 or an analog thereof as an active ingredient.

IV. Specific Disclosures of the Present Invention

(1) Antiviral Agents that Reduce or Inhibit the Activity of SR Proteins

(i) Antiviral Agents that Act by Dephosphorylating SR Proteins

The antiviral agents that act by dephosphorylating SR proteins includeactivators that activate Phosphatase 2A (Mumby, M. C. and Walter, G.(1993) Physiol. Rev. 73, 673-680; Lechward, K., Awotunde, O. S.,Swiatek, W. and Muszynska, G. (2001) Acta Biochim. Pol. 48, 921-933;Cohen, P. (1989) The structure and regulation of protein phosphatases.Annu. Rev. Biochem. 58, 453-508; Janssens, V. and Goris, J. (2001)Biochem. J. 353, 417-39). Specifically, such antiviral agents includepolypeptides encoded by HIV tat gene (for example, accession numberAAK08486), polypeptides encoded by adenovirus E4-ORF4 (for example,accession number AAB37507), or polypeptides encoded by vaccinia virusVH1 (for example, accession number AAV38329). Furthermore, suchantiviral agents also include expression vectors for gene therapy, whichcarry a HIV tat gene, adenovirus E4-ORF4 gene, or vaccinia virus VH1gene. A tat gene is available, for example, as CDS (nucleotides5830-6044 plus nucleotides 8369-8411) under accession number AF324493;E4-ORF4 is available, for example, as CDS (nucleotides 1634-1993) underaccession number 582508; vaccinia virus VH1 is available, for example,as CDS (nucleotides 1-555) under accession number BT019522.

(ii) SR Protein Kinase Inhibitors

(ii-1)

There are various kinases already known as enzymes that phosphorylate SRproteins, but these enzymes are thought to phosphorylate RS domains atdifferent sites. The present inventors discovered that SRPKs are theonly RS kinases that achieve the specific phosphorylation whichcontributes to SR protein stabilization. Thus, to prevent thestabilization of SR proteins through phosphorylation, the target SRprotein kinases particularly include SRPKs. The SRPKs include both SRPK1(Nature (1994) Vol. 369, pp. 678-682) and SRPK2 (Biochem. Biophys. Res.Commun. (1998) Vol. 242: pp. 357-364; Wang, H. Y. et al., J. Cell. Biol.1998, 140:737-750). The nucleotide sequence of SRPK1 gene is set forth,for example, in nucleotides 124-2088, nucleotides 109-2073, nucleotides10-2487, and nucleotides 43-1986 of accession numbers NM_(—)003137,U09564, AJ318054, and NM_(—)016795, respectively. The amino acidsequence is set forth, for example, in accession numbers NP_(—)003128,AAA20530, CAC39299, CAA11833, and NP_(—)058075. Meanwhile, thenucleotide sequence of SRPK2 gene is set forth, for example, innucleotides188-2245 and nucleotides 208-2253 of accession numbers U88666and NM_(—)009274, respectively. The amino acid sequence is set forth,for example, in AAC05299 and NP_(—)033300 (Nikolakaki, E. et al., J.Biol. Chem. 276, 40175-40182 (2001); Papoutsopoulou, S., et al., NucleicAcids Res. 27, 2972-2980 (1999); Wang, H. Y. et al., Genomics 57,310-315 (1999); Gui, J. F. et al., Nature 369, 678-682 (1994); Wang, H.Y. et al., J. Cell Biol. 140, 737-750 (1998); Papoutsopoulou, S. et al.,Nucleic Acids Res. 27, 2972-2980 (1999); Kuroyanagi, N. et al., Biochem.Biophys. Res. Commun. 242, 357-364 (1998); Bedford, M. T. et al., EMBOJ. 16, 2376-2383 (1997)). SRPK1s also include the species referred to as“SRPK 1a”.

Substances having the function of inhibiting kinases (SRPKs), which areused in the methods of the present invention, include compounds(including SRPIN-1 and analogs thereof) represented by the followingformula:

and pharmaceutically acceptable salts and hydrates thereof;

-   wherein, R¹ represents a hydrogen atom, a C₁₋₆ alkyl group which may    have substituents, a C₂₋₆ alkenyl group which may have substituents,    a C₂₋₆ alkynyl group which may have substituents, a C₆₋₁₀ aryl group    which may have substituents, a halogen atom, a nitro group, a cyano    group, an azide group, a hydroxy group, a C₁₋₆ alkoxy group which    may have substituents, a C₁₋₆ alkylthio group which may have    substituents, a C₁₋₆ alkylsulfonyl group which may have    substituents, a carboxyl group, a formyl group, a C₁₋₆    alkoxycarbonyl group which may have substituents, an acyl group, an    acylamino group, or a sulfamoyl group;-   R² represents a hydrogen atom, a C₁₋₆ alkyl group which may have    substituents or an aryl group which may have substituents;-   R³ represents a C₁₋₆ alkyl group which may have substituents, a C₂₋₆    alkenyl group which may have substituents, a C₆₋₁₀ aryl group which    may have substituents, a nitrogen-containing heterocycle which may    have substituents, or a condensed aromatic heterocycle which may    have substituents;-   R⁴ represents a hydrogen atom or a halogen atom;-   Q represents —C(O)—, —C(S)—, —SO₂—, —C(S)NHC(O)—, —C(O)NHC(O)—, or    —C(O)NHC(S)—;-   W represents a hydrogen atom, a C₁₋₆ alkyl group which may have    substituents, a C₆₋₁₀ aryl group which may have substituents, a    halogen atom, a hydroxy group, a C₁₋₆ alkoxy group which may have    substituents, a C₁₋₆ alkylthio group which may have substituents, a    nitrogen-containing heterocycle which may have substituents, a    condensed aromatic heterocycle which may have substituents, or a    group represented by the following formula (II);

-   -   wherein, R⁵ and R⁶ are the same or different and each represent        a hydrogen atom, a C₁₋₆ alkyl group which may have substituents,        a nitrogen-containing heterocycle which may have substituents, a        condensed aromatic heterocycle which may have substituents, an        acyl group, or an acylamino group; or    -   the above R⁵ and R⁶, together with the adjacent nitrogen atom,        may form a heterocycle which may have substituents, and the        heterocycle may be a condensed aromatic heterocycle which may        have substituents;    -   the above R⁵ and R⁶ may be a cycloalkylidene amino group which        may have substituents or an aromatic condensed cycloalkylidene        group which may have substituents.

Examples of the compounds described above include the compoundsrepresented by the following formula:

X includes F, Cl, Br, I, and At.

Specifically, such compounds includes SRPIN-1, represented by thefollowing formula:

The SRPIN-1 of the present invention is available from Maybridge(Trevillett, Tintagel, Cornwall PL34 OHW, England) and Ambinter (46 quaiLouis Bleriot, Paris, F-75016 France); however, the following outlinesits chemical synthesis:

-   (ii-2) Antiviral agents using RNAi targeting SRPK1 gene and SRPK2    gene

Inside cells, siRNAs, morpholino oligos, or miRNAs can be used to reducethe expression level of genes encoding SRPK1 and SRPK2.

Known methods can be used to design siRNAs. The RNAs may be designed,for example, by the following methods:

-   (ii-2-1)

The sequences that can be used as siRNA targets avoid the 5′ and 3′ UTRs(untranslated region) and sequences adjacent to the start codon; are 50nucleotides or more downstream of the start codon; are within the ORFand start from AA or NA; comprise 19 to 21 nucleotides (most typically19 nucleotides) whose CG content is about 50%; and minimal sequencebiases and repeats at the 5′ and 3′ ends.

When the target sequence starts with AA, siRNAs may be prepared tocomprise a dinucleotide overhang of dTdT or UU. Alternatively, when thetarget sequence starts with NA, siRNAs may be prepared to comprise dTdN,dTdT, or UU.

To prevent cross-reactions with sequences other than the target sequencefrom affecting expression of proteins other than the target protein, aBLAST search or the like will confirm whether the selected sequence hashomology with other RNA sequences.

The present invention also includes embodiments that use siRNAexpression vectors, constructed to allow intracellular expression of thedesigned siRNAs.

The morpholino oligos are compounds in which multiplenucleotide-comprising morpholino subunits are linked in a chaincomprising structures that link the morpholine ring and non-ionicphosphorodiamidate subunits (U.S. Pat. Nos. 5,142,047; 5,185,444). Sincemorpholino antisense oligos are highly stable in cells and have highaffinity for mRNAs, they can thus be preferably used to inhibit theexpression of target genes (Summerton J E., Ann NY Acad Sci 2003; 1002:189). Methods for designing effective morpholino oligos are alreadyknown (see Summerton, 1989, In: Discoveries in Antisense Nucleic Acids;Ed.: C. Brakel; Pub.: The Portfolio Publishing Co., Woodlands, Texas;pages 71-80; Summerton & Weller, 1997, Antisense Nuc. Acid Drug Dev. 7,187; and the Gene Tools website). Morpholino oligos are available fromGene Tools (Gene Tools, LLC, Philomath, Oreg.).

(2) The Antiviral Agents that Act by Inhibiting the Expression of theGenes Encoding SR Proteins Include, For Example, siRNAs, MorpholinoOligos, and miRNAs.

-   (2-1) siRNAs

siRNAs can be designed using the methods described above in (ii-2).

(3) Antiviral Agents Comprising Proteins that Antagonize SR Proteins orthat Act by Activating These Proteins

-   (3-1)

The phrase “antagonize SR protein” means promoting the selection of a 3′splice site distal to an intron in splicing, for example. Specifically,the activity of SR proteins can be canceled using a splicing regulatoryfactor that antagonizes SR proteins, which promote the selection of 3′splice sites proximal to an intron. Specifically, such proteins thatantagonize SR proteins include heteronuclear ribonucleoproteins(hnRNPs), such as hnRNP A1, A2, and B1, but hnRNP A1 is preferable. Morepreferable are antiviral agents that are gene therapy expression vectorscarrying an hnRNP A1-encoding gene. The gene sequence encoding hnRNP A1is set forth, for example, in nucleotides 105-1064 and nucleotides105-1220 of accession numbers NM_(—)002136 and NM_(—)031157,respectively. The amino acid sequence of hnRNP A1 is set forth inaccessions number NP_(—)002127 and NP_(—)112420, and others(Expert-Bezan, Sureau, A. et al., J. Biol. Chem. 279, 38249-38259(2004); Zahler, A. M. et al., J. Biol. Chem. 279, 10077-10084 (2004);Marchand, V. et al., J. Mol. Biol. 323, 629-652 (2002); Buvoli, M. etal., EMBO J. 9, 1229-1235 (1990); Biamonti, G. et al., J. Mol. Biol.207, 491-503 (1989); Buvoli, M. et al., Nucleic Acids Res. 16, 3751-3770(1988); Michael, W. M. et al., Cell 83, 415-422 (1995)). The genesequence encoding hnRNP A2/B1 is set forth, for example, in nucleotides170-1192 and nucleotides 170-1228 of accession numbers NM_(—)002137 andNM_(—)031243, respectively. The amino acid sequence of hnRNP A2/B 1 isset forth in accession numbers NP_(—)002128 and NP_(—)112533, and others(Kozu, T., et al., Genomics 25, 365-371 (1995); Biamonti, G. et al.,Nucleic Acids Res. 22, 1996-2002 (1994); Burd, C. G. et al., Proc. Natl.Acad. Sci. U.S.A. 86, 9788-9792 (1989); Kumar, A. et al., J. Biol. Chem.261, 11266-11273 (1986)).

(4) Methods for Screening for Antiviral Agents, Which Comprise Screeningfor Substances that Inhibit SRPKs

The methods for screening for antiviral agents of the present inventionare, for example, methods comprising the selection of SRPK inhibitors,which comprise the steps of reacting test compounds with SRPKs, andtesting the ability of the SRPKs to phosphorylate SR proteins. Compoundsthat impair this ability (SRPK inhibitors) are useful as antiviralagents. In particular, the present invention also includes methods forscreening for antiviral agents, which comprise screening variouscompounds that target SRPK1 or SRPK2 for SRPK inhibitors, using SRproteins or peptides with two or more consecutive units of RS or SR asSRPK substrates. Compounds that inhibit the SR protein-phosphorylatingactivity of SRPKs can be selected efficiently by using peptides with twoor more consecutive units of Arg-Ser (RS) or Ser-Arg (SR) as SRPKsubstrates, and selecting compounds that impair SRPK's ability tophosphorylate the substrates (SRPK inhibitors).

More specifically, the screenings of the present invention comprise thesteps of:

-   (a) contacting an SRPK with a substrate in the presence of a test    compound;-   (b) detecting the phosphorylation of the substrate; and-   (c) selecting compounds that impair phosphorylation compared to when    the test compound is absent or present in small amounts.

As described above, the substrates include SR proteins, partialpolypeptides thereof which comprise RS domains, and polypeptides withtwo or more consecutive units of RS or SR (see the Examples). The SRPKsmay be wild type SRPK1 or SRPK2. Alternatively, the SRPKs may be fusionproteins comprising tag peptides or other modified proteins, as long asthey retain phosphorylation activity. Herein, SRPKs comprising mutationsor such are also referred to as “SRPK”, as long as they retain theactivity of phosphorylating SR proteins.

More specifically, herein, SRPK1 includes:

-   (a) a protein comprising an amino acid sequence of accession numbers    NP_(—)003128, AAA20530, CAC39299, CAA11833, or NP_(—)058075;-   (b) a protein with phosphorylation activity which comprises an amino    acid sequence that exhibits 80% or higher sequence identity,    preferably 85% or higher sequence identity, more preferably 90% or    higher sequence identity, still more preferably 95% or higher    sequence identity to this amino acid sequence;-   (c) a protein with phosphorylation activity which is encoded by a    nucleic acid which hybridizes under stringent conditions to a    complementary strand of a nucleic acid comprising the whole or a    portion of nucleotides 124-2088 of accession number NM_(—)003137,    nucleotides 109-2073 of accession number U09564, nucleotides 10-2487    of accession number AJ318054, or nucleotides 43-1986 of accession    number NM_(—)016795. Herein, SRPK2 includes:-   (a) a protein comprising an amino acid sequence of accession numbers    AAC05299 or NP_(—)033300;-   (b) a protein with phosphorylation activity comprising an amino acid    sequence that exhibits 80% or higher sequence identity, preferably    85% or higher sequence identity, more preferably 90% or higher    sequence identity, still more preferably 95% or higher sequence    identity to this amino acid sequence;-   (c) a protein with phosphorylation activity which is encoded by a    nucleic acid which hybridizes under stringent conditions to a    complementary strand of a nucleic acid comprising the whole or a    portion of nucleotides 188-2245 of accession number U88666, or    nucleotides 208-2253 of accession number NM_(—)009274. The “portion”    means, for example, 20 or more consecutive nucleotides, preferably    25 or more nucleotides, more preferably 30 or more nucleotides, 40    or more nucleotides, 45 or more nucleotides, 50 or more nucleotides.

Amino acid sequence identity can be determined, for example, using theBLASTP program (Altschul, S. F. et al., 1990, J. Mol. Biol. 215:403-410). For example, homology searches are carried out at the BLASTwebpage of NCBI (National Center for Biotechnology Information) usingdefault parameters with all filters, including Low complexity, switchedoff (Altschul, S. F. et al. (1993) Nature Genet. 3:266-272; Madden, T.L. et al. (1996) Meth. Enzymol. 266:131-141; Altschul, S. F. et al.(1997) Nucleic Acids Res. 25:3389-3402; Zhang, J. & Madden, T. L. (1997)Genome Res. 7:649-656). Parameters may be set, for example, as follows:gap open cost=11, gap extend cost=1, wordsize=2, Dropoff (X) for blastextensions in bits=7, X dropoff value for gapped alignment (in bits)=15,final X dropoff value for gapped alignment (in bits)=25. BLOSUM62 isused as a score matrix. Sequence identity can be determined, forexample, by aligning two sequences using the blast2 sequences program,which compares two sequences (Tatiana A et al. (1999) FEMS MicrobiolLett. 174:247-250). Gaps are treated in the same way as mismatches. Anidentity score is calculated for the entire amino acid sequence of thewild type protein, which is set forth in the above accession numbers(for example, the entire sequence of SEQ ID NO: 2 or 4). Identity scoresmay be calculated disregarding gaps outside the amino acid sequence ofthe wild type protein in the alignment. For hybridization, a probe isprepared from either a nucleic acid comprising the coding sequence ofthe wild type protein (for example, SEQ ID NO: 1 or 3) or a nucleic acidtargeted in the hybridization, and whether or not the probe willhybridize to other nucleic acids can be identified by detection.Stringent hybridization conditions include, for example, conditionswhere hybridization is carried out using a solution containing 5×SSC(1×SSC comprises 150 mM NaCl and 15 mM sodium citrate), 7% (W/V) SDS, 10μg/ml denatured salmon sperm DNA, and 5× Denhardt's solution (1×Denhardt's solution contains 0.2% polyvinylpyrrolidone, 0.2% bovineserum albumin, and 0.2% Ficoll) at 48° C., preferably at 50° C., andmore preferably at 52° C., and where post-hybridization washing iscarried out for two hours at the same temperature as in thehybridization, more preferably at 60° C., still more preferably at 65°C., and even more preferably at 68° C. using 2×SSC, preferably 1×SSC,more preferably 0.5×SSC, and still more preferably 0.1×SSC, withshaking.

The phosphorylation activity of SRPKs can be detected, for example, byconducting a reaction between an SRPK and a substrate using labeled ATP,and quantifying the labeled substrate. Specifically, the methodsdescribed in Example 4B can be followed.

Compounds exhibiting marked antiviral activity may be further selectedfrom the yielded compounds by detecting antiviral activity through theadditional steps of:

-   (d) detecting viral propagation or the expression of a viral gene in    the presence of a selected test compound; and-   (e) selecting a compound that reduces viral propagation or viral    gene expression compared to when the compound is absent or present    in small amounts.

As described in the Examples, viral propagation or viral gene expressioncan be evaluated, for example, by detecting the production of viralproteins in cells introduced with the viral genome.

The present invention also relates to SRPK inhibitors and antiviralagents that comprise compounds selected by the above-described screeningmethods of the present invention. The present invention also relates touses of the compounds obtained by the above-described screening methodsof the present invention for producing SRPK inhibitors and/or antiviralagents, and uses of the same in SRPK inhibition and/or antiviraltreatments. For example, compounds selected from the group consisting ofthe compounds of CAS Registry Nos. 218156-96-8, 674360-18-0,494830-83-0, 672919-05-0, 54231-51-5, 10338-55-3, 1692-79-1, 1496-40-81,496012-09-0, 445406-05-3, 445412-62-4, and 388071-30-5 are useful asSRPK inhibitors and/or antiviral agents.

The present invention also includes uses of the above antiviral agentsas viral propagation inhibitors or antiviral therapeutic agents. Forexample, when SRPIN-1 is used as an antiviral agent, in addition toSRPIN-1, known pharmaceutical adjuvants, for example, AZT and proteaseinhibitors, may be added.

The viral propagation inhibitors or antiviral therapeutic agents of thepresent invention may be administered, for example, orally,percutaneously, submucosally, subcutaneously, intramuscularly,intravascularly, intracerebrally, or intraperitoneally, intermittentlyor continuously so that their concentration in the body falls within therange of 100 nM to 1 mM.

The SRPIN-1 analog compounds of the present invention are described inmore detail below. The present invention relates to compounds with thestructure indicated below, and to uses thereof.

Compounds of the present invention are aniline derivatives representedby the following formula (I):

or pharmaceutically acceptable salts or hydrates thereof;

-   wherein, R¹ represents a hydrogen atom, a C₁₋₆ alkyl group which may    have substituents, a C₂₋₆ alkenyl group which may have substituents,    a C₂₋₆ alkynyl group which may have substituents, a C₆₋₁₀ aryl group    which may have substituents, a halogen atom, a nitro group, a cyano    group, an azide group, a hydroxy group, a C₁₋₆ alkoxy group which    may have substituents, a C₁₋₆ alkylthio group which may have    substituents, a C₁₋₆ alkylsulfonyl group which may have    substituents, a carboxyl group, a formyl group, a C₁₋₆    alkoxycarbonyl group which may have substituents, an acyl group, an    acylamino group, or a sulfamoyl group;-   R² represents a hydrogen atom, a C₁₋₆ alkyl group which may have    substituents, or an aryl group which may have substituents;-   R³ represents a C₁₋₆ alkyl group which may have substituents, a C₂₋₆    alkenyl group which may have substituents, a C₆₋₁₀ aryl group which    may have substituents, a nitrogen-containing heterocycle which may    have substituents, or a condensed aromatic heterocycle which may    have substituents;-   R⁴ represents a hydrogen atom or a halogen atom;-   Q represents —C(O)—, —C(S)—, —SO₂—, —C(S)NHC(O)—, —C(O)NHC(O)—, or    —C(O)NHC(S)—;-   W represents a hydrogen atom, a C₁₋₆ alkyl group which may have    substituents, a C₆₋₁₀ aryl group which may have substituents, a    halogen atom, a hydroxy group, a C₁₋₆ alkoxy group which may have    substituents, a C₁₋₆ alkylthio group which may have substituents, a    nitrogen-containing heterocycle which may have substituents, a    condensed aromatic heterocycle which may have substituents, or a    group represented by the following formula (II):

-   -   wherein, R⁵ and R⁶ are the same or different and each represent        a hydrogen atom, a C₁₋₆ alkyl group which may have substituents,        a nitrogen-containing heterocycle which may have substituents, a        condensed aromatic heterocycle which may have substituents, an        acyl group, or an acylamino group; or    -   the above R⁵ and R⁶, together with the adjacent nitrogen atom,        may form a heterocycle which may have substituents, and the        heterocycle may be a condensed aromatic heterocyclic group which        may have substituents;    -   the above R⁵ and R⁶ may be a cycloalkylidene amino group which        may have substituents, or an aromatic condensed cycloalkylidene        group which may have substituents.

Among such compounds represented by formula (I), preferable compoundsinclude, for example, the following compounds:

-   (1) compounds in which the above R¹ is a hydrogen atom, a C₁₋₆ alkyl    group which may have substituents, or a halogen atom;-   (2) compounds in which the above R¹ is a hydrogen atom, a C₁₋₆ alkyl    group, a halogenated C₁₋₆ alkyl group, or a halogen atom;-   (3) compounds in which the above R¹ is a hydrogen atom, a methyl    group, a trifluoromethyl group, a chlorine atom, or a fluorine atom;-   (4) compounds in which the above R¹ is a hydrogen atom or a    trifluoromethyl group;-   (5) compounds in which the above R² is a hydrogen atom or a C₁₋₆    alkyl group;-   (6) compounds in which the above R² is a hydrogen atom or a methyl    group;-   (7) compounds in which the above R² is a hydrogen atom;-   (8) compounds in which the above R³ is a C₆₋₁₀ aryl group which may    have substituents or a nitrogen-containing 5- to 10-membered    heteroaryl ring which may have substituents;-   (9) compounds in which the above R³ is a phenyl group; C₆₋₁₀ aryl    group which has as a substituent a C₁₋₆ alkyl group, a C₁₋₆ alkoxy    group, or a nitro group; or a nitrogen-containing 5- to 10-membered    heteroaryl group;-   (10) compounds in which the above R³ is a phenyl group; a phenyl    group which has as a substituent a C₁₋₆ alkyl group, a C₁₋₆ alkoxy    group, or a nitro group; or a pyridyl group;-   (11) compounds in which the above R³ is a phenyl group, a tolyl    group, a methoxyphenyl group, a nitrophenyl group, or a pyridyl    group;-   (12) compounds in which the above R³ is a tolyl group or a pyridyl    group;-   (13) compounds in which the above R³ is a 4-pyridyl group;-   (14) compounds in which the above R⁴ is a hydrogen atom;-   (15) compounds in which the above Q is —C(O)— or —C(O)NHC(S)—, where    C(O) means that an oxygen atom is linked with a carbon atom via a    double bond, and C(S) means that a sulfur atom is linked with a    carbon atom via a double bond;-   (16) compounds in which the above Q is —C(O)—;-   (17) compounds in which the above W is a hydrogen atom, a halogen    atom, or a group represented by the following formula (II):

wherein, R⁵ and R⁶ are the same or different and each represent a C1-6alkyl group which may have substituents; or

-   the above R⁵ and R⁶, together with the adjacent nitrogen atom, may    form a heterocyclic group which may have substituents; and the    heterocyclic group may be a condensed aromatic heterocyclic group    which may have substituents;-   (18) compounds in which the above W is a 4- to 8-membered    heterocyclic group having one nitrogen atom, which may have a C₁₋₆    alkyl group as a substituent, a 4- to 8-membered heterocyclic group    comprising one nitrogen atom and one oxygen atom, which may have a    C₁₋₆ alkyl group as a substituent, or a 4- to 8-membered    heterocyclic group which is condensed with a phenyl group and    comprises one nitrogen atom;-   (19) compounds in which the above W is a 4- to 8-membered    heterocyclic group comprising one nitrogen atom, which may have a    C₁₋₆ alkyl group as a substituent;-   (20) compounds in which the above W is a piperidinyl group or a    perhydroazepine group, which may have a C₁₋₆ alkyl group as a    substituent; and-   (21) compounds in which the above W is a hydrogen atom, a halogen    atom, a diethylamino group, a pyrrolidinyl group, a piperidinyl    group, a 2-methylpiperidinyl group, a perhydroazepine group, an    indolinyl group, an isoindolinyl group, or a    1,2,3,4-tetrahydroquinolyl group.

In the compounds described above, R¹ is preferred in the order of (1) to(4), with (4) most preferred. R² is more preferred in the order of (5)to (7), with (7) most preferred. R³ is more preferred in the order of(8) to (13), with (13) most preferred. Q is more preferred in the orderof (15) to (16), with (16) most preferred. W is more preferred in theorder of (17) to (20), with (20) most preferred. W defined in (21) isalso preferred.

More preferable compounds are represented by the above formula (I), andcomprise arbitrary combinations of preferable substituent types, each ofwhich is selected from the group consisting of (1) to (4), the groupconsisting of (5) to (7), the group consisting of (8) to (13), the groupconsisting of (14), the group consisting of (15) to (16), or the groupconsisting of (17) to (21).

Specific compounds represented by formula (I) are shown herein below,but the present invention is not to be construed as being limitedthereto.

TABLE 1 Compound No. Structural Formula 340

341

342

343

344

345

346

347

348

349

TABLE 2 Compound No. Structural Formula 608

609

610

611

612

613

614

615

616

617

TABLE 3 Compound No. Structural Formula 618

619

620

621

622

623

624

625

626

The present invention relates to any of the compounds shown as examplesabove, but of these compounds, preferable compounds are those ofCompound Nos. 340, 341, 342, 343, 344, 345, 346, 347, 348, 608, 613,615, 616, 618, 619, 620, 621, 622, 623, 624, 625, and 626; morepreferable are the compounds of Compound Nos. 340, 341, 342, 343, 345,347, 348, 608, 613, 615, 616, 618, 619, 620, 622, 623, 624, 625, and626; still more preferable are the compounds of Compound Nos. 340, 348,613, 616, 618, 622, and 624; and further more preferable are thecompounds of Compound Nos. 340, 348, 613, 618, and 624.

The present invention also relates to any of the compounds shown aboveas examples. In particular, the present invention also relates to novelcompounds selected from the group consisting of the compounds ofCompound Nos. 341, 342, 346, 347, 348, 349, 612, 613, 614, 616, 617,618, 619, 620, 621, 622, and 624, which are shown above as examples.

These compounds (aniline derivatives), or pharmaceutically acceptablesalts or hydrates thereof, are effective as SRPK inhibitors.

The compounds (aniline derivatives), or pharmaceutically acceptablesalts or hydrates thereof, are also useful as antiviral agents.

Representative methods for producing the compounds of the presentinvention, represented by the above formula (I), are described below.

The R¹, R², R³, R⁴, R⁵, R⁶, Q, and W below are defined as above. Roomtemperature means a temperature ranging from about 20 to 30° C.

Production Method A

Step 1

In this step, compound 1a is reacted with compound 2a to give compound3a. The material “nitrobenzene derivative 1a” may be availablecommercially or by appropriately inducing functional groups. X is ahalogen atom or sulfonate used as a leaving group. Compound 2a is areagent comprising the —NR⁵R⁶ to be introduced. It is preferable to useone to two equivalents of compound 2a. The reaction may be conducted ina solvent in the presence of a base.

It is possible to use triethylamine, diisopropyl ethylamine, pyridine,4-(dimethylamino)pyridine, or such as the base. It is preferable to useone to five equivalents of base. Alternatively, an excess amount (one tofive equivalents) of H—NR⁵R⁶ may be used as the base.

The solvents include, for example, dimethyl sulfoxide,N,N-dimethylformamide, N-methylpyrrolidone, dioxane, tetrahydrofuran,and toluene.

The reaction temperature range from 0° C. to 150° C. However, roomtemperature is preferable.

Step 2

In this step, the nitro group of compound 3a is reduced to an aminogroup to give compound 4a.

The reduction method can be to contact concentrated hydrochloric acid orsuch in the presence of tin chloride or such in the solvent.Alternatively, standard reduction reactions, such as catalytichydrogenation, can also be used.

The reaction solvents include methanol, ethanol, N,N-dimethylformamide,tetrahydrofuran, 1,2-dimethoxyethane, 1,4-dioxane, and water, and mixedsolvents comprising combinations thereof.

It is preferable to use a mass ratio of 1 to 20 equivalents of tinchloride or such, as a reducing agent. The reaction can be conducted ata temperature ranging from 0° C. to 100° C.

Compounds 3a and 4a may sometimes be available commercially, and in thiscase commercially available products may be used. In particular, when Win formula (I) is hydrogen or a halogen, the compound is usuallycommercially available. For example, the compounds of Compound Nos. 608,612, and 623 to 626 shown herein are included in such compounds.

Step 3a

In this step, compound 4a is reacted with compound 5a to give compound6a. L represents a halogen atom or such. The reaction can be conductedin a solvent in the presence of a base, and in the presence of acatalyst if required. It is preferable to use one to three equivalentsof compound 5a in this reaction.

The reaction solvents include dichloromethane, chloroform, 1,4-dioxane,tetrahydrofuran, toluene, pyridine, N,N-dimethylformamide,N-methylpyrrolidone, and the like.

As the base, triethylamine, diisopropyl ethylamine, pyridine,4-(dimethylamino)pyridine, and such may be used.

Standard amide bond-forming reactions using condensing agents can beused when L is a hydroxyl group, and standard amide bond-formingreactions can also be used when L is a leaving group, such as asuccinimidyl group or imidazole group.

The catalysts include 4-(dimethylamino) pyridine and such.

The reaction temperature may range from 0° C. to 100° C.

Step 3b

In this step, compound 4a is reacted with compound 5b to give compound6b.

The reaction can be conducted using acyl isothiocyanate in a solvent inthe presence of a base. Acyl isothiocyanate may be commerciallyavailable, or may be prepared by reacting an appropriate acyl halide andthiocyanate in solution, and then used as is. It is preferable to useone to five equivalents of acyl isothiocyanate. The thiocyanates thatcan be used include potassium thiocyanate, sodium thiocyanate, andammonium thiocyanate. One to five equivalents of thiocyanate arepreferably used.

The solvents include, for example, acetonitrile, N,N-dimethylformamide,N-methylpyrrolidone, tetrahydrofuran, ethylene glycol dimethyl ether,and 1,4-dioxane.

The bases include, for example, triethylamine, disopropyl ethylamine,pyridine, and 4-(dimethylamino) pyridine. It is preferable to use one tofive equivalents of the base.

The reaction can be conducted at a temperature ranging from 0° C. to150° C.

Step 4a

In this step, the amide group of compound 6a is alkylated (convertedinto R²) to give compound 7a.

The reaction can be conducted in a solvent using an alkylating agent(R²—X) in the presence of a base. X is a halogen atom or sulfonate whichserves as a leaving group. One to five equivalents of the alkylatingagent (R²—X) are preferably used.

The solvents include, for example, N,N-dimethylformamide,N-methylpyrrolidone, tetrahydrofuran, ethylene glycol dimethyl ether,1,4-dioxane, acetonitrile, and ether.

The bases include sodium hydride, potassium hydride, lithium hydride,butyl lithium, methyl lithium, phenyl lithium, and lithiumdiisopropylamide. One to five equivalents of base are preferably used.

The reaction can be conducted at a temperature ranging from 0° C. to150° C.

Step 4b

In this step, the carbonyl group with an amide bond in compound 6a isconverted into a thiocarbonyl group to give compound 7b.

The reaction is conducted using a thiocarbonylating agent in a solvent.The thiocarbonylating agents include, for example, Lawesson's reagent(2,4-bis(4-methoxyphenyl)-1,3,2,4-dithiadiphosphetane 2,4-disulfide) andphosphorous pentasulfide (phosphorus decasulfide, P₄S₁₀). It ispreferable to use one to five equivalents of thiocarbonylating agent.

The solvents include, for example, toluene, benzene, chlorobenzene,xylene, N,N-dimethylformamide, N-methylpyrrolidone, ethylene glycoldimethyl ether, 1,4-dioxane, and tetrahydrofuran.

The reaction can be conducted at a temperature ranging from 0° C. to200° C.

The above are representative methods for producing compound (I) of thepresent invention. The material compounds and various reagents used toproduce the compounds of the present invention may form salts, hydrates,or solvates thereof, and each vary depending on the type of startingmaterials or solvents and such to be used; they are not particularlylimited as long as they do not inhibit the reaction. The types ofsolvents used varies with the types of starting materials and reagentsand such. Of course, the solvents are not particularly limited as longas they dissolve the starting material to some extent and do not inhibitthe reaction. When compound (I) of the present invention is yielded in afree form, it can be converted according to conventional methods into asalt or hydrate thereof that may be formed by compound (I).

When compound (I) of the present invention is yielded as a salt or ahydrate thereof, it can be converted into a free form of the abovecompound (I) according to conventional methods.

Various isomers (for example, geometric isomers, optical isomers basedon asymmetric carbons, rotational isomers, stereoisomers, and tautomers)of compound (I) of the present invention can be purified and isolatedusing conventional isolation means, for example, recrystallization,diastereomer salt methods, enzyme-based resolution methods, variouschromatographic methods (for example, thin-layer chromatography, columnchromatography, and gas chromatography).

In the present invention, SRPIN-1 analogs can be used to inhibit theactivity of SRPKs. Specifically, the phosphorylation activity of SRPK1and/or SRPK2 can be inhibited by administering the SRPIN-1 analogsdescribed herein. The present invention relates to uses of SRPIN-1analogs to inhibit SRPK activity. The present invention also relates toSRPK inhibitors comprising SRPIN-1 analogs. The present invention alsorelates to uses of SRPIN-1 analogs to produce SRPK inhibitors.Furthermore, the present invention also relates to methods forinhibiting SRPK activity, which comprises the step of contacting anSRPIN-1 analog with a SRPK. The phrase “contacting with SRPK” can meanthat an SRPIN-1 analog is administered in vitro or in vivo to cells,tissues, and/or individuals expressing SRPK.

In the present invention, SRPIN-1 analogs can also be used to inhibitviral propagation. Specifically, viral propagation is inhibited when thephosphorylation activity of SRPK1 and/or SRPK2 is inhibited byadministering the SRPIN-1 analogs described herein. The presentinvention relates to uses of SRPIN-1 analogs to inhibit viralpropagation. The present invention also relates to antiviral agentscomprising SRPIN-1 analogs. The present invention also relates to usesof SRPIN-1 analogs to produce antiviral agents. The present inventionalso relates to methods for inhibiting viral propagation, which comprisethe step of contacting an SRPIN-1 analog with a SRPK. The phrase“contacting with SRPK” can mean that an SRPIN-1 analog is administeredin vitro or in vivo to cells, tissues, and/or individuals expressingSRPK.

The present invention also provides packages comprising theabove-descried SRPIN-1 analogs or pharmaceutically acceptable salts orhydrates thereof, where the fact that the compounds have SRPK-inhibitingand/or antiviral activity is recorded on the package or packagecontents. Herein a package refers to a package that contains an SRPIN-1analog, or pharmaceutically acceptable salt or hydrate thereof. Thepackages may include a container for the SRPIN-1 analog orpharmaceutically acceptable salt or hydrate thereof, and may furtherinclude a bag or outer case or such to contain the container.

The present invention also provides packages comprising compounds thatreduce the activity or expression level of SR proteins, where the factthat the compounds have antiviral activity is recorded on the package orpackage contents. In particular, the present invention provides packagesin which the compound is a compound having the activity of inhibitingthe expression and/or activity of an SRPK.

The compounds of the present invention can be formulated intocompositions in combination with pharmaceutically acceptable carriers.For example, the compounds may be formulated into pharmaceuticalcompositions using known preparation techniques. When the pharmaceuticalcompositions of the present invention are used as SRPK inhibitors,antiviral agents (specifically, preventive or therapeutic agents forviral diseases), or other pharmaceuticals, they can be administered, forexample, orally in dosage forms, such as tablets, capsules, granules,powders, pills, troches, or syrups, or parenterally in dosage forms,such as injections, aerosols, suppositories, patches, poultices,lotions, liniments, ointments, or eye drops. Such preparations areproduced by known methods using additives, such as excipients,lubricants, binders, disintegrating agents, stabilizers, flavoringagents, and diluents.

Excipients include, for example, starches, such as starch, potatostarch,and cornstarch; lactate; crystalline cellulose; and calcium hydrogenphosphate.

Coating agents include, for example, ethyl cellulose, hydroxypropylcellulose, hydroxypropylmethyl cellulose, shellac, talc, carnauba wax,and paraffin.

Binders include, for example, polyvinylpyrrolidone, Macrogol, and thesame compounds as described above for the excipients.

Disintegrating agents include, for example, the same compounds asdescribed above for the excipients; and chemically modified starches andcelluloses, such as cross carmellose sodium, carboxymethyl starchsodium, and cross-linked polyvinylpyrrolidone.

Stabilizers include, for example, paraoxybenzoates such as methylparabenand propylparaben; alcohols such as chlorobutanol, benzylalcohol, andphenyl ethyl alcohol; benzalkonium chloride; phenols such as phenol andcresol; thimerosal; dehydroacetic acid; and sorbic acid.

Flavoring agents include, for example, generally used sweeteners,acidifiers, and spices.

Solvents used to produce solutions include ethanol, phenol,chlorocresol, purified water, and distilled water.

Detergents and emulsifiers include, for example, polysorbate 80,polyoxyl 40 stearate, and Lauromacrogol.

When the pharmaceutical compositions of the present invention are usedas SRPK inhibitors or antiviral agents, the doses of the compound of thepresent invention or the pharmaceutical acceptable salts thereof arevaried depending on the symptoms, age, type of administration procedure,and such. For example, depending on the symptoms, when administeredorally the compounds are preferably administered at a daily dose of 0.01mg (preferably 0.1 mg) (lower limit) to 2000 mg (preferably 500 mg, morepreferably 100 mg) (upper limit) per patient (warm-blooded animals,human in particular), administered at one time or divided into severaltimes. When administered intravenously, the compounds are preferablyadministered at a daily dose of 0.001 mg (preferably 0.01 mg) (lowerlimit) to 500 mg (preferably 50 mg) (upper limit) per adult,administered at one time or divided into several times, depending on thesymptoms.

EXAMPLES

Herein below, the present invention will be specifically described usingExamples, however, it is not to be construed as being limited thereto.All publications cited herein have been incorporated as parts of thisdescription.

Silica gel (MERCK 9385-5B, 70-230 mesh) was used in columnchromatography as described below. Thin-layer chromatography (TLC) wascarried out using glass plates pre-coated with silica gel (MERCK 5715,silica gel 60 F₂₅₄). Melting points were measured using a Yanaco MP-500Dmicro melting point apparatus, manufactured by Yanaco AnalyticalInstruments Corp. ¹H NMR spectra were measured using a NMR spectrometerJNM AL-400 manufactured by JEOL Ltd. CDCl₃ or CD₃OD (ISOTEC) was used asa solvent in the measurement of NMR spectra. Chemical shift is expressedas a relative value when tetramethylsilane ((CH₃)₄Si) is used as aninternal standard (0 ppm). The coupling constant (J) is shown in Hz. Thesymbols, s, d, t, m, and br, represent singlet, doublet, triplet,qualtet, multiplet, and broad peak, respectively.

Referential Example 1 Synthesis of SRPIN-1

Representative synthesis methods for SRPIN-1 (code name GIF-0340) aredescribed below.

Referential Example 1-1A

Piperidine (220 μl, 2.22 mnol) and N,N-diisopropylethylamine (220 μl,2.40 mmol) were sequentially added at room temperature to anN,N-dimethylformamide (DMF; 1 ml) solution containing1-fluoro-2-nitro-4-(trifluoromethyl)benzene (427 mg, 2.04 mmol,commercially available product). The resulting mixture was stirred forone hour. Water was added to the mixture, and the resulting mixture wasextracted three times with ether. The extracted organic layer was washedwith brine, dried over Na₂SO₄, filtered, and concentrated under reducedpressure.

The residue was purified by silica gel column chromatography (40 g,hexane/ethyl acetate=10/1). Thus,1-[2-nitro-4-(trifluoromethyl)phenyl]piperidine (561 mg, 2.04 mmol,quant.) was yielded as an orange-colored solid.

The results of TLC and ¹H NMR (CDCl₃, 400 MHz) are as follows: TLC R_(f)0.47 (hexane/acetone=16/1); ¹H NMR (CDCl₃, 400 MHz) δ 1.61-1.68 (m, 2H,CH₂), 1.72 (tt, 4H, J=5.3, 5.3 Hz, 2CH₂), 3.13 (t, 4H, J=5.3 Hz, 2CH₂),7.13 (d, 1H, J=8.8 Hz, aromatic) 7.61 (dd, 1H, J=2.0, 8.8 Hz, aromatic),8.03 (d, 1H, J=2.0 Hz, aromatic).

Referential Example 1-2A

Concentrated hydrochloric acid (2.00 ml, 24.0 mmol) and anhydrous tindichloride (2.50 g, 13.1 mmol) were sequentially added at 0° C. to amethanol (10 ml) solution containing1-[2-nitro-4-(trifluoromethyl)phenyl]piperidine (559 mg, 2.03 mmol),obtained as described in Referential Example 1-1A. The resulting mixturewas warmed to room temperature and then stirred for 17.5 hours. Asaturated aqueous solution of sodium bicarbonate was added to themixture. The resulting mixture was extracted three times with ethylacetate. The obtained organic layer was washed with brine, dried overNa₂SO₄, filtered, and concentrated under reduced pressure. The residuewas purified by silica gel column chromatography (50 g, hexane/ethylacetate=14/1)). Thus, 2-(1-piperidinyl)-5-(trifluoromethyl)aniline (448mg, 1.83 mmol, 90.4%) was yielded as a pale yellow solid.

The results of TLC and ¹H NMR (CDCl₃, 400 MHz) are as follows: TLC R_(f)0.30(hexane/acetone=18/1); ¹H NMR (CDCl₃, 400 MHz) δ 1.59-1.60 (m, 2H,CH₂), 1.71 (tt, 4H, J=5.4, 5.4 Hz, 2CH₂), 2.85 (brs, 4H, 2CH₂), 4.09(brs, 2H, NH₂), 6.92 (d, 1H, J=1.9 Hz, aromatic), 6.97 (dd, 1H, J=1.9,8.4 Hz, aromatic), 7.01 (d, 1H, J=8.4 Hz, aromatic).

Referential Example 1-3A

Isonicotinoyl chloride hydrochloride (151 mg, 0.850 mmol, commerciallyavailable product), triethylamine (450 μl, 3.23 mmol), and a catalyticamount of 4-(dimethylamino)pyridine were sequentially added at 0° C. toa dichloromethane (5 ml) solution of2-(1-piperidinyl)-5-(trifluoromethyl)aniline (173 mg, 0.708 mmol),obtained as described in Referential Example 1-2A. The resulting mixturewas warmed to room temperature and stirred for 19.5 hours. Water wasadded to the mixture, and the resulting mixture was extracted threetimes with ethyl acetate. The obtained organic layer was washed with asaturated aqueous solution of sodium bicarbonate, dried over Na₂SO₄,filtered, and concentrated under reduced pressure. The residue waspurified by silica gel column chromatography (10 g, hexane/ethylacetate=1.5/1) and recrystallization (hexane). Thus,N-[2-(1-piperidinyl)-5-(trifluoromethyl)phenyl]isonicotinamide (SRPIN-1,code name GIF-0340) (83.8 mg, 0.240 mmol, 33.9%) was yielded as acolorless solid.

The melting point, and results of TLC and ¹H NMR (CDCl₃, 400 MHz), areas follows: m.p. 96-98° C.; TLC R_(f) 0.40 (hexane/ethyl acetate=1/1);¹H NMR (CDCl₃, 400 MHz) δ 1.67-1.68 (m, 2H, CH₂), 1.78 (tt, 4H, J=5.5,5.5 Hz, 2CH₂), 2.88 (t, 4H, J=5.5 Hz, 2CH₂), 7.29 (d, 1H, J=8.2 Hz,aromatic), 7.40 (dd, 1H, J=1.8, 8.2 Hz, aromatic), 7.76 (dd, 2H, J=2.0,4.4 Hz, aromatic), 8.86 (dd, 2H, J=2.0, 4.4 Hz, aromatic), 8.87 (d, 1H,J=1.8 Hz, aromatic), 9.53 (s, 1H, NH).

Referential Example 1-1B

Piperidine (5.50 ml, 55.5 mmol, commercially available product) wasadded at 0° C. to an N,N-dimethylformamide (DMF; 7 ml) solution of1-chloro-2-nitro-4-(trifluoromethyl)benzene (5.00 g, 22.4 mmol,commercially available product). The resulting mixture was stirred for40 minutes. Water was added to the mixture, and the resulting mixturewas extracted three times with ethyl acetate. The obtained organic layerwas washed with a saturated sodium chloride solution, dried overanhydrous sodium sulfate, filtered, and concentrated under reducedpressure. The residue was purified by silica gel column chromatography(200 g, hexane/ethyl acetate=8/1). Thus,1-[2-nitro-4-(trifluoromethyl)phenyl]piperidine (6.13 g, quant.) wasyielded as an orange-colored solid.

Referential Example 1-2B

Concentrated hydrochloric acid (12.2 ml, 146 mmol) and anhydrous tindichloride (12.7 g, 67.2 mmol) were sequentially added at 0° C. to adichloromethane solution (10 ml) of1-[2-nitro-4-(trifluoromethyl)phenyl]piperidine (6.13 g, 22.4 mmol),obtained as described in Referential Example 1-1B. The resulting mixturewas stirred for seven hours. Water was added to the mixture, and theresulting mixture was extracted three times with ethyl acetate. Theobtained organic layer was washed with a saturated sodium chloridesolution, dried over anhydrous sodium sulfate, filtered, andconcentrated under reduced pressure. The residue was purified by silicagel column chromatography (200 g, hexane/ethyl acetate=15/1). Thus,2-(1-piperidinyl)-5-(trifluoromethyl)aniline (4.55 g, 83.0%) was yieldedas a pale yellow solid.

Referential Example 1-3B

Isonicotinoyl chloride hydrochloride (6.48 g, 36.4 mmol, commerciallyavailable product) and triethylamine (5.57 ml, 54.6 mmol) weresequentially added at 0° C. to a dichloromethane (10 ml) solution of2-(1-piperidinyl)-5-(trifluoromethyl)aniline (4.45 g, 18.2 mmol)obtained as described in Referential Example 1-2B. The mixture wasstirred for half an hour. Water was added to the mixture, and theresulting mixture was extracted three times with ethyl acetate. Theobtained organic layer was washed with a saturated sodium chloridesolution, dried over anhydrous sodium sulfate, filtered, andconcentrated under reduced pressure. The residue was purified by silicagel column chromatography (200 g, hexane/ethyl acetate=1/1) andrecrystallization. Thus, N-[2-(1-piperidinyl)-5-(trifluoromethyl)phenyl]isonicotinamide (SRPIN-1, GIF-0340) (5.49 g, 86.3%) was yieldedas a colorless solid.

Referential Example 2 Synthesis of Code Name GIF-0613

Referential Example 2-1

(S)-2-methylpiperidine (270 μl, 2.24 mmol, commercially availableproduct) was added at room temperature to an N,N-dimethylformamide (DMF;0.5 ml) solution of 1-fluoro-2-nitro-4-(trifluoromethyl)benzene (211 mg,1.00 mmol, commercially available product). The resulting mixture wasstirred for two hours. Water was added to the mixture, and the resultingmixture was extracted three times with ethyl acetate. The obtainedorganic layer was washed with a saturated sodium chloride solution,dried over anhydrous sodium sulfate, filtered, and concentrated underreduced pressure. The residue was purified by silica gel columnchromatography (20 g, hexane/ethyl acetate=12/1). Thus,(S)-1-[2-nitro-4-(trifluoromethyl)phenyl]-2-methylpiperidine (286 mg,99.2%) was yielded as an orange-colored oily material.

TLC R_(f) 0.44 (hexane/ethyl acetate=16/1).

Referential Example 2-2

Concentrated hydrochloric acid (1.00 ml, 12.0 mmol) and anhydrous tindichloride (903 mg, 4.76 mmol) were sequentially added at 0° C. to amethanol (5 ml) solution of(S)-1-[2-nitro-4-(trifluoromethyl)phenyl]-2-methylpiperidine (275 mg,0.953 mmol), obtained as described in [Referential Example 2-1]. Theresulting mixture was warmed to room temperature and stirred for 17hours. A saturated solution of sodium hydrogen carbonate was added tothe mixture, and the resulting mixture was extracted three times withethyl acetate. The obtained organic layer was washed with a saturatedsodium chloride solution, dried over anhydrous sodium sulfate, filtered,and concentrated under reduced pressure. The residue was purified bysilica gel column chromatography (50 g, hexane/ethyl acetate=12/1).Thus, (S)-2-(2-methyl-1-piperidinyl)-5-(trifluoromethyl)aniline (233 mg,94.6%) was yielded as a colorless oily material.

TLC R_(f) 0.38 (hexane/ethyl acetate=16/1).

Referential Example 2-3

Isonicotinoyl chloride hydrochloride (466 mg, 2.61 mmol, commerciallyavailable product) and triethylamine (600 μl, 4.30 mmol) weresequentially added at 0° C. to a dichloromethane (5 ml) solution of(S)-2-(2-methyl-1-piperidinyl)-5-(trifluoromethyl)aniline (223 mg, 0.863mmol), obtained as described in [Referential Example 2-2]. The resultingmixture was warmed to room temperature and stirred for 19.5 hours. Waterwas added to the mixture, and the resulting mixture was extracted threetimes with ethyl acetate. The obtained organic layer was washed with asaturated sodium chloride solution, dried over anhydrous sodium sulfate,filtered, and concentrated under reduced pressure. The residue waspurified by silica gel column chromatography (25 g, hexane/ethylacetate=1.5/1). Thus,(S)—N-[2-(2-methyl-1-piperidinyl)-5-(trifluoromethyl)phenyl]isonicotinamide(GIF-0613) (293 mg, 93.4%) was yielded as an colorless oily material.

The results of TLC and ¹H NMR (CDCl₃, 400 MHz) are as follows: TLC R_(f)0.40 (hexane/ethyl acetate=1/1); ¹H NMR (CDCl₃, 400 MHz) δ 0.84 (d, 3H,J=6.4 Hz, CH₃), 1.39-1.69 (m, 3H, CH₂, CH), 1.82-1.86 (m, 1H, CH),1.92-1.95 (m, 2H, CH₂), 2.65-2.72 (m, 1H, CH), 2.89-2.92 (m, 1H, CH),2.98-3.02 (m, 1H, CH), 7.35 (d, 1H, J=8.4 Hz, aromatic), 7.40 (dd, 1H,J=2.2, 8.4 Hz, aromatic), 7.75 (dd, 2H, J=1.8, 4.4 Hz, aromatic), 8.86(dd, 2H , J=1.8, 4.4 Hz, aromatic), 8.93 (d, 1H, J=1.8 Hz, aromatic),10.1 (s, 1H, NH).

Referential Example 3 Synthesis of Code Name GIF-0617

Referential Example 3-1

Pyrrolidine ((983 μl, 12.0 mmol, commercially available product) wasadded at 0° C. to a N,N-dimethylformamide (DMF; 4 ml) solution of1-fluoro-2-nitro-4-(trifluoromethyl)benzene (1.02 g, 4.89 mmol,commercially available product). The resulting mixture was warmed toroom temperature and stirred for 4.5 hours. Water was added to themixture, and the resulting mixture was extracted three times with ethylacetate. The obtained organic layer was washed with a saturated sodiumchloride solution, dried over anhydrous sodium sulfate, filtered, andconcentrated under reduced pressure. The residue was purified by silicagel column chromatography (50 g, hexane/ethyl acetate=5/1). Thus,1-[2-nitro-4-(trifluoromethyl)phenyl]pyrrolidine (1.26 g, 99.3%) wasyielded as an orange-colored solid.

TLC R_(f) 0.45 (hexane/ethyl acetate=5/1).

Referential Example 3-2

Concentrated hydrochloric acid (1.36 ml, 16.3 mmol) and anhydrous tindichloride (1.55 g, 8.16 mmol) were sequentially added at 0° C. to amethanol (4 ml) solution of1-[2-nitro-4-(trifluoromethyl)phenyl]pyrrolidine (606 mg, 2.33 mmol),obtained as described in Referential Example 3-1. The resulting mixturewas stirred for four hours. A saturated solution of sodium hydrogencarbonate was added to the mixture, and the resulting mixture wasextracted three times with ethyl acetate. The obtained organic layer waswashed with a saturated sodium chloride solution, dried over anhydroussodium sulfate, filtered, and concentrated under reduced pressure. Theresidue was purified by silica gel column chromatography (50 g,hexane/ethyl acetate=15/1). Thus,1-[2-amino-4-(trifluoromethyl)phenyl]pyrrolidine (550 mg, quant.) wasyielded as a red-orange colored oily material.

TLC R_(f) 0.63 (hexane/ethyl acetate=1/1).

Referential Example 3-3

Isonicotinoyl chloride hydrochloride (705 mg, 3.96 mmol, commerciallyavailable product) and triethylamine (823 μl, 5.94 mmol) weresequentially added at 0° C. to a dichloromethane (10 ml) solution of1-[2-amino-4-(trifluoromethyl)phenyl]pyrrolidine (516 mg, 2.24 mmol),obtained as described in Referential Example 3-2. The resulting mixturewas warmed to room temperature and stirred for five hours. Water wasadded to the mixture, and the resulting mixture was extracted threetimes with dichloromethane. The obtained organic layer was washed with asaturated sodium chloride solution, dried over anhydrous sodium sulfate,filtered, and concentrated under reduced pressure. The residue waspurified by silica gel column chromatography (25 g, hexane/ethylacetate=1/2). Thus,N-[2-(1-pyrrolidinyl)-5-(trifluoromethyl)phenyl]isonicotinamide(GIF-0617) (734 mg, 97.8%) was yielded as a colorless solid.

The melting point, and results of TLC and ¹H NMR (CDCl₃, 400 MHz), areas follows: m.p. 134-135° C.; TLC R_(f) 0.29 (hexane/ethyl acetate=1/1);¹H NMR (CDCl₃, 400 MHz) δ 2.01 (tt, 4H, J=3.2 Hz, 6.4 Hz, 2CH₂), 3.15(t, 4H, J=6.4 Hz, 2CH₂), 7.19 (d, 1H, J=8.5 Hz, aromatic), 7.38 (dd, 1H,J=2.2, 8.5 Hz, aromatic), 7.71 (dd, 2H, J=1.6, 4.4 Hz, aromatic), 8.53(d, 1H, J=2.2, Hz, aromatic), 8.79 (s, 1H, NH), 8.83 (dd, 2H, J=1.6, 4.4Hz, aromatic).

Referential Example 4 Synthesis of Code Name GIF-0618

Referential Example 4-1

Hexahydro-1H-azepine (682 μl, 6.05 mmol, commercially available product)was added at 0° C. to an N,N-dimethylformamide (DMF; 2 ml) solution of1-fluoro-2-nitro-4-(trifluoromethyl)benzene (506 mg, 2.42 mmol,commercially available product). The resulting mixture was warmed toroom temperature and stirred for one hour. Water was added to themixture, and the resulting mixture was extracted three times with ethylacetate. The obtained organic layer was washed with a saturated sodiumchloride solution, dried over anhydrous sodium sulfate, filtered, andconcentrated under reduced pressure. The residue was purified by silicagel column chromatography (20 g, hexane/ethyl acetate=7/1). Thus,hexahydro-1-[2-nitro-4-(trifluoromethyl)phenyl]-1H-azepine (680 mg,97.5%) was yielded as an orange-colored solid.

The results of TLC and ¹H NMR (CDCl₃, 400 MHz) are as follows: TLC R_(f)0.49 (hexane/ethyl acetate=5/1); ¹H NMR (CDCl₃, 400 MHz) δ 1.57-1.63 (m,4H, 2CH₂), 1.79-1.83 (m, 4H, 2CH₂), 3.31 (t, 4H, J=5.5 Hz, 2CH₂), 7.11(d, 1H, J=9.1 Hz, aromatic) 7.53 (dd, 1H, J=2.0, 9.1 Hz, aromatic), 7.99(d, 1H, J=2.0 Hz, aromatic).

Referential Example 4-2

Concentrated hydrochloric acid (1.27 ml, 15.2 mmol) and anhydrous tindichloride (1.43 g, 7.54 mmol) were sequentially added at 0° C. to amethanol (5 ml) solution ofhexahydro-1-[2-nitro-4-(trifluoromethyl)phenyl]-1H-azepine (675 mg, 2.34mmol), obtained as described in Referential Example 4-1. The resultingmixture was stirred for two hours. A saturated solution of sodiumhydrogen carbonate was added to the mixture, and the resulting mixturewas extracted three times with ethyl acetate. The obtained organic layerwas washed with a saturated sodium chloride solution, dried overanhydrous sodium sulfate, filtered, and concentrated under reducedpressure. The residue was purified by silica gel column chromatography(30 g, hexane/ethyl acetate=20/1). Thus,1-[2-amino-4-(trifluoromethyl)phenyl]-hexahydro-1H-azepine (522 mg,86.3%) was yielded as a colorless solid.

The results of TLC and ¹H NMR (CDCl₃, 400 MHz) are as follows: TLC R_(f)0.81 (hexane/ethyl acetate=3/1); ¹H NMR (CDCl₃, 400 MHz) δ 1.70-1.84 (m,8H, 4CH₂), 3.04 (t, 4H, J=5.4, Hz, 2CH₂), 4.10 (brs, 2H, NH₂), 6.92 (d,1H, J=1.2 Hz, aromatic), 6.94 (dd, 1H, J=1.2, 7.9 Hz, aromatic), 7.05(d, 1H, J=7.9 Hz, aromatic).

Referential Example 4-3

Isonicotinoyl chloride hydrochloride (704 mg, 3.95 mmol, commerciallyavailable product) and triethylamine (823 μl, 5.94 mmol) weresequentially added at 0° C. to a dichloromethane (6 ml) solution of1-[2-amino-4-(trifluoromethyl)phenyl]-hexahydro-1H-azepine (512 mg, 1.98mmol), obtained as described in Referential Example 4-2. The resultingmixture was stirred for one and a half hours. Water was added to themixture, and the resulting mixture was extracted three times withdichloromethane. The obtained organic layer was washed with a saturatedsodium chloride solution, dried over anhydrous sodium sulfate, filtered,and concentrated under reduced pressure. The residue was purified bysilica gel column chromatography (30 g, hexane/ethyl acetate=2/1). Thus,N-[2-(1-hexahydro-1H-azepinyl)-5-(trifluoromethyl)phenyl]isonicotinamide(GIF-0618) (697 mg, 97.0%) was yielded as a colorless solid.

The melting point, and results of TLC and ¹H NMR (CDCl₃, 400 MHz), areas follows: m.p. 138-139° C.; TLC R_(f) 0.40 (hexane/ethyl acetate=1/1);¹H NMR (CDCl₃, 400 MHz) δ 1.79 (br, 8H, 4CH₂), 3.06-3.10 (m, 4H, 2CH₂),7.31 (d, 1H, J=8.2 Hz, aromatic), 7.37 (dd, 1H, J=1.6, 8.2 Hz,aromatic), 7.76 (dd, 2H, J=2.0, 6.0 Hz, aromatic), 8.85 (m, 3H,aromatic), 9.66 (s, 1H, NH).

Referential Example 5 Synthesis of Code Name GIF-0346

Referential Example 5-1

Morpholine (190 μl, 2.17 mmol, commercially available product) was addedat room temperature to an N,N-dimethylformamide (DMF; 0.5 ml) solutionof 1-fluoro-2-nitro-4-(trifluoromethyl)benzene (209 mg, 1.00 mmol,commercially available product). The resulting mixture was stirred forthree hours. Water was added to the mixture, and the resulting mixturewas extracted three times with ethyl acetate. The obtained organic layerwas washed with a saturated sodium chloride solution, dried overanhydrous sodium sulfate, filtered, and concentrated under reducedpressure. The residue was purified by silica gel column chromatography(20 g, hexane/ethyl acetate=3/1). Thus,4-[2-nitro-4-(trifluoromethyl)phenyl]morpholine (270 mg, 97.7%) wasyielded as an orange-colored oily material.

TLC R_(f) 0.27 (hexane/ethyl acetate=3/1).

Referential Example 5-2

Concentrated hydrochloric acid (1.00 ml, 12.0 mmol) and anhydrous tindichloride (905 mg, 4.77 mmol) were sequentially added at 0° C. to amethanol (5 ml) solution of4-[2-nitro-4-(trifluoromethyl)phenyl]morpholine (263 mg, 0.952 mmol),obtained as described in Referential Example 5-1. The resulting mixturewas warmed to room temperature and stirred for 20 hours. A saturatedsolution of sodium hydrogen carbonate was added to the mixture, and theresulting mixture was extracted three times with ethyl acetate. Theobtained organic layer was washed with a saturated sodium chloridesolution, dried over anhydrous sodium sulfate, filtered, andconcentrated under reduced pressure. The residue was purified by silicagel column chromatography (20 g, hexane/ethyl acetate=2/1). Thus,4-[2-amino-4-(trifluoromethyl)phenyl]morpholine (214 mg, 91.2%) wasyielded as a colorless solid.

TLC R_(f) 0.31 (hexane/ethyl acetate=3/1).

Referential Example 5-3

Isonicotinoyl chloride hydrochloride (320 mg, 1.80 mmol, commerciallyavailable product) and triethylamine (480 μl, 3.44 mmol) weresequentially added at 0° C. to a dichloromethane (5 ml) solution of4-[2-amino-4-(trifluoromethyl)phenyl]morpholine (196 mg, 0.796 mmol),obtained as described in Referential Example 5-2. The resulting mixturewas warmed to room temperature and stirred for 60 hours. Water was addedto the mixture, and the resulting mixture was extracted three times withdichloromethane. The obtained organic layer was washed with a saturatedsodium chloride solution, dried over anhydrous sodium sulfate, filtered,and concentrated under reduced pressure. The residue was purified bysilica gel column chromatography (30 g, hexane/ethyl acetate=2/1). Thus,N-[2-(4-morpholinly)-5-(trifluoromethyl)phenyl]isonicotinamide(GIF-0346) (65.1 mg, 23.2%) was yielded as a colorless solid.

The melting point, and results of TLC and ¹H NMR (CDCl₃, 400 MHz), areas follows: m.p. 172-173° C.; TLC R_(f) 0.23 (hexane/ethyl acetate=1/3);¹H NMR (CDCl₃, 400 MHz) δ 2.96 (t, 4H, J=4.4 Hz, 2CH₂), 3.92 (t, 4H,J=4.4 Hz, 2CH₂), 7.34 (d, 1H, J=8.4 Hz, aromatic), 7.44 (dd, 1H, J=1.6,8.4 Hz, aromatic), 7.75 (dd, 1H, J=1.6, 4.4 Hz, aromatic), 8.87-8.88 (m,3H , aromatic) 9.48 (s, 1H, NH).

Referential Example 6 Synthesis of Code Name GIF-0347

Referential Example 6-1

Diethylamine (230 μl, 2.22 mmol, commercially available product) wasadded at room temperature to an N,N-dimethylformamide (DMF; 0.5 ml)solution of 1-fluoro-2-nitro-4-(trifluoromethyl)benzene (211 mg, 1.01mmol, commercially available product). The resulting mixture was stirredfor three hours. Water was added to the mixture, and the resultingmixture was extracted three times with ethyl acetate. The obtainedorganic layer was washed with a saturated sodium chloride solution,dried over anhydrous sodium sulfate, filtered, and concentrated underreduced pressure. The residue was purified by silica gel columnchromatography (20 g, hexane/ethyl acetate=8/1). Thus,1-diethylamino-2-nitro-4-(trifluoromethyl)benzene (258 mg, 98.3%) wasyielded as an orange-colored oily material.

TLC R_(f) 0.37 (hexane/ethyl acetate/ether =16/1/1).

Referential Example 6-2

Concentrated hydrochloric acid (1.00 ml, 12.0 mmol) and anhydrous tindichloride (908 mg, 4.78 mmol) were sequentially added at 0° C. to amethanol (5 ml) solution of1-diethylamino-2-nitro-4-(trifluoromethyl)benzene (251 mg, 0.957 mmol),obtained as described in Referential Example 6-1. The resulting mixturewas warmed to room temperature and stirred for 22 hours. A saturatedsolution of sodium hydrogen carbonate was added to the mixture, and theresulting mixture was extracted three times with ethyl acetate. Theobtained organic layer was washed with a saturated sodium chloridesolution, dried over anhydrous sodium sulfate, filtered, andconcentrated under reduced pressure. The residue was purified by silicagel column chromatography (20 g, hexane/ethyl acetate=20/1-10/1). Thus,2-amino-1-diethylamino-4-(trifluoromethyl)benzene (144 mg, 64.7%) wasyielded as a colorless oily material.

TLC R_(f) 0.22 (hexane/ethyl acetate=30/1).

Referential Example 6-3

Isonicotinoyl chloride hydrochloride (88.2 mg, 0.495 mmol, commerciallyavailable product) and triethylamine (140 μL, 1.00 mmol) weresequentially added at 0° C. to a dichloromethane (3 ml) solution of2-amino-1-diethylamino-4-(trifluoromethyl)benzene (103 mg, 0.443 mmol),obtained as described in Referential Example 6-2. The resulting mixturewas warmed to room temperature and stirred for one and a half hours.Water was added to the mixture, and the resulting mixture was extractedthree times with dichloromethane. The obtained organic layer was washedwith a saturated sodium chloride solution, dried over anhydrous sodiumsulfate, filtered, and concentrated under reduced pressure. The residuewas purified by silica gel column chromatography (30 g, hexane/ethylacetate=2/1). Thus,N-[2-diethylamino-5-(trifluoromethyl)phenyl]isonicotinamide (GIF-0347)(51.0 mg, 34.1%) was yielded as a colorless solid.

The melting point, and results of TLC and ¹H NMR (CDCl₃, 400 MHz), areas follows: m.p. 78-80° C.; TLC R_(f) 0.31 (hexane/ethyl acetate=1/1);¹H NMR (CDCl₃, 400 MHz) δ 1.01 (t, 6H, J=7.1 Hz, 2CH₃), 3.04 (q, 4H,J=7.1 Hz, 2CH₂), 7.33 (d, 1H, J=8.2 Hz, aromatic), 7.41 (dd, 1H, J=1.8,8.2 Hz, aromatic), 7.73 (dd, 2H, J=1.8, 4.4 Hz, aromatic), 8.60 (d, 1H,J=2.6 Hz, aromatic), 8.85 (dd, 2H, J=1.8, 4.4 Hz, aromatic), 8.91 (d,1H, J=1.8 Hz, aromatic) 9.90 (s, 1H, NH).

Referential Example 7 Synthesis of Code Name GIF-0343

Nicotinoyl chloride hydrochloride (122 mg, 0.685 mmol, commerciallyavailable product) and triethylamine (250 μl, 1.79 mmol) weresequentially added at 0° C. to a dichloromethane (5 ml) solution of2-(1-piperidinyl)-5-(trifluoromethyl)aniline (152 mg, 0.622 mmol),obtained as described in Referential Example 1-2. The resulting mixturewas warmed to room temperature and stirred for 16.5 hours. Water wasadded to the mixture, and the resulting mixture was extracted threetimes with ethyl acetate. The obtained organic layer was washed with asaturated sodium chloride solution, dried over anhydrous sodium sulfate,filtered, and concentrated under reduced pressure. The residue waspurified by silica gel column chromatography (15 g, hexane/ethylacetate=1.5/1-1/1). Thus,N-[2-(1-piperidinyl)-5-(trifluoromethyl)phenyl]nicotinamide (GIF-0343)(98.4 mg, 45.3%) was yielded as a colorless solid.

The melting point, and results of TLC and ¹H NMR (CDCl₃, 400 MHz), areas follows: m.p. 145-146° C.; TLC R_(f) 0.40 (hexane/ethyl acetate=1/1);¹H NMR (CDCl₃, 400 MHz) δ 1.62-1.69 (m, 2H, CH₂), 1.79 (tt, 4H, J=5.8,5.8 Hz, 2CH₂), 2.88 (t, 4H, J=5.8 Hz, 2CH₂), 7.29 (d, 1H, J=8.4 Hz,aromatic), 7.39 (dd, 1H, J=2.0, 8.4 Hz, aromatic), 7.51 (dd, 1H, J=4.8,8.0 Hz, aromatic), 8.30 (ddd, 1H, J=1.6, 2.4, 8.0 Hz, aromatic), 8.82(dd, 1H, J=1.6, 4.8 Hz, aromatic), 8.87 (d, 1H, J=2.0 Hz, aromatic),9.16 (d, 1H, J=2.4 Hz, aromatic), 9.53 (s, 1H, NH).

Referential Example 8 Synthesis of Code Name GIF-0344

Benzoyl chloride (50.0 μl, 0.430 mmol, commercially available product)and triethylamine (170 μl, 1.21 mmol) were sequentially added at 0° C.to a dichloromethane (3 ml) solution of2-(1-piperidinyl)-5-(trifluoromethyl)aniline (102 mg, 0.417 mmol),obtained as described in Referential Example 1-2. The resulting mixturewas warmed to room temperature and stirred for 18 hours. Water was addedto the mixture, and the resulting mixture was extracted three times withethyl acetate. The obtained organic layer was washed with a saturatedsodium chloride solution, dried over anhydrous sodium sulfate, filtered,and concentrated under reduced pressure. The residue was purified bysilica gel column chromatography (20 g, hexane/ethyl acetate=10/1).Thus, N-[2-(1-piperidinyl)-5-(trifluoromethyl)phenyl]benzamide(GIF-0344) (126 mg, 86.7%) was yielded as a colorless solid.

The melting point, and results of TLC and ¹H NMR (CDCl₃, 400 MHz), areas follows: m.p. 128-129° C.; TLC R_(f) 0.46 (hexane/ethyl acetate=4/1);¹H NMR (CDCl₃, 400 MHz) δ 1.62-1.70 (m, 2H, CH₂), 1.78 (tt, 4H, J=5.2,5.2 Hz, 2CH₂), 2.88 (t, 4H, J=5.2 Hz, 2CH₂), 7.26 (d, 1H, J=8.6 Hz,aromatic), 7.35 (d, 1H, J=0.8, 8.6 Hz, aromatic), 7.52-7.61 (m, 1H,aromatic), 8.10 (m, 2H, aromatic), 7.94 (m, 2H, aromatic), 8.91 (d, 1H,J=0.8 Hz, aromatic), 9.44 (s, 1H, NH).

Referential Example 9 Code Name GIF-0345

4-Nitrobenzoyl chloride (64.2 mg, 0.345 mmol, commercially availableproduct) and triethylamine (120 μl, 0.859 mmol) were sequentially addedat 0° C. to a dichloromethane (2 ml) solution of2-(1-piperidinyl)-5-(trifluoromethyl)aniline (51.2 mg, 0.209 mmol),obtained as described in Referential Example 1-2. The resulting mixturewas warmed to room temperature and stirred for 60 hours. Water was addedto the mixture, and the resulting mixture was extracted three times withethyl acetate. The obtained organic layer was washed with a saturatedsodium chloride solution, dried over anhydrous sodium sulfate, filtered,and concentrated under reduced pressure. The residue was purified bysilica gel column chromatography (30 g, hexane/ethyl acetate=8/1-6/1).Thus, N-[2-(1-piperidinyl)-5-(trifluoromethyl)phenyl]-4-nitrobenzamide(GIF-0345) (46.3 mg, 56.3%) was yielded as a yellow solid.

The melting point, and results of TLC and ¹H NMR (CDCl₃, 400 MHz), areas follows: m.p. 125-136° C.; TLC R_(f) 0.33 (hexane/ethyl acetate=4/1);¹H NMR (CDCl₃, 400 MHz) δ 1.62-1.70 (m, 2H, CH₂), 1.77 (tt, 4H, J=5.0,5.0 Hz, 2CH₂), 2.88 (t, 4H, J=5.0 Hz, 2CH₂), 7.30 (d, 1H, J=8.0 Hz,aromatic), 7.40 (dd, 1H, J=1.6, 8.2 Hz, aromatic), 8.10 (dd, 2H, J=1.8,6.8 Hz, aromatic), 8.41 (d, 2H, J=1.8, 6.8 Hz, aromatic), 8.86 (d, 1H,J=2.0 Hz, aromatic), 9.54 (s, 1H, NH).

Referential Example 10 Synthesis of Code Name GIF-0615

4-Methoxybenzoyl chloride (250 mg, 1.47 mmol, commercially availableproduct) and triethylamine (254 μl, 1.83 mmol) were sequentially addedat 0° C. to a dichloromethane (4 ml) solution of2-(1-piperidinyl)-5-(trifluoromethyl)aniline (147 mg, 0.602 mmol),obtained as described in Referential Example 1-2. The resulting mixturewas warmed to room temperature and stirred for 17 hours. Water was addedto the mixture, and the resulting mixture was extracted three times withethyl acetate. The obtained organic layer was washed with a saturatedsodium chloride solution, dried over anhydrous sodium sulfate, filtered,and concentrated under reduced pressure. The residue was purified bysilica gel column chromatography (20 g, hexane/ethyl acetate=5/1). Thus,N-[2-(1-piperidinyl)-5-(trifluoromethyl)phenyl]-4-methoxybenzamide(GIF-0615) (240 mg, quant.) was yielded as a colorless solid.

The melting point, and results of TLC and ¹H NMR (CDCl₃, 400 MHz), areas follows: m.p. 111-114° C.; TLC R_(f) 0.33 (hexane/ethyl acetate=4/1);¹H NMR (CDCl₃, 400 MHz) δ 1.64-1.68 (m, 2H, CH₂), 1.78 (tt, 4H, J=5.4,5.4 Hz, 2CH₂), 1.60-1.70 (m, 2H, CH₂), 2.39-2.41 (m, 2H, CH₂), 2.87 (t,4H, J=5.4 Hz, 2CH₂), 3.89 (s, 3H, CH₃), 7.03 (dd, 2H, J=2.0, 7.0 Hz,aromatic), 7.23 (d, 1H, J=8.0 Hz, aromatic), 7.32 (dd, 1H, J=1.6, 8.0Hz, aromatic), 7.91 (dd, 2H, J=2.0, 7.0 Hz, aromatic), 8.88 (d, 1H,J=1.6 Hz, aromatic), 9.34 (s, 1H, NH).

Referential Example 11 Synthesis of Code Name GIF-0622

p-Toluenesulfonyl chloride (233 g, 1.22 mmol, commercially availableproduct) and triethylamine (254 μl, 1.83 mmol) were sequentially addedat room temperature to a dichloromethane (5 ml) solution of2-(1-piperidinyl)-5-(trifluoromethyl)aniline (149 mg, 0.610 mmol),obtained as described in Referential Example 1-2. The resulting mixturewas stirred for 60 hours. Water was added to the mixture, and theresulting mixture was extracted three times with ethyl acetate. Theobtained organic layer was washed with a saturated sodium chloridesolution, dried over anhydrous sodium sulfate, filtered, andconcentrated under reduced pressure. The residue was purified by silicagel column chromatography (20 g, hexane/ethyl acetate=10/1). Thus, N-[2-(1 -piperidinyl)-5-(trifluoromethyl)phenyl]-p-toluenesulfonamide(GIF-0622) (243 mg, quant) was yielded as a colorless solid.

The melting point, and results of TLC and ¹H NMR (CDCl₃, 400 MHz), areas follows: m.p. 117-134° C.; TLC R_(f) 0.49 (hexane/ethyl acetate=5/1);¹H NMR (CDCl₃, 400 MHz) δ 1.55-1.60 (m, 2H, CH₂), 1.66 (tt, 4H, J=5.2,5.2 Hz, 2CH₂), 2.36 (s, 3H, CH₃), 2.51 (t, 4H, J=5.2 Hz, 2CH₂), 7.11 (d,1H, J=8.0 Hz, aromatic), 7.22-7.28 (m, 3H, aromatic), 7.71 (dd, 2H,J=1.8, 8.6 Hz, aromatic), 7.85 (d, 1H, J=2.0 Hz, aromatic), 7.94 (s, 1H,NH).

Referential Example 12 Synthesis of Code Name GIF-0624

An acetonitrile (15 ml) solution containing potassium thiocyanate (119mg, 1.22 mmol, commercially available product) and nicotinoyl chloridehydrochloride (352 mg, 1.97 mmol, commercially available product) wasstirred at 70° C. for 40 minutes. The mixture was cooled to roomtemperature, and then an acetonitrile (5 ml) solution of2-(1-piperidinyl)-5-(trifluoromethyl)aniline (244 mg, 1.00 mmol),obtained as described in Referential Example 1-2, and triethylamine (278μl, 2.00 mmol) were sequentially added thereto. The resulting mixturewas stirred at 50° C. for one hour. Water was added to the mixture, andthe resulting mixture was extracted three times with dichloromethane.The obtained organic layer was washed with a saturated sodium chloridesolution, dried over anhydrous sodium sulfate, filtered, andconcentrated under reduced pressure. The residue was purified by silicagel column chromatography (25 g, hexane/ethyl acetate=4/1-1/1). Thus,1-nicotinoyl-3-[2-(1-piperidinyl)-5-(trifluoromethyl)phenyl]thiourea(GIF-0624) (383 mg, 93.7%) was yielded as a pale yellow solid.

The melting point, and results of TLC and ¹H NMR (CD₃OD, 400 MHz), areas follows: m.p. 142-144° C.; TLC R_(f) 0.26 (hexane/ethyl acetate=1/1);¹H NMR (CDCl₃, 400 MHz) δ 1.58-1.67 (m, 2H, CH₂), 1.75-1.85 (m, 4H,2CH₂), 2.89-2.95 (m, 4H, 2CH₂), 7.33-7.40 (m, 1H, aromatic), 7.44-7.48(m, 1H, aromatic), 7.60-7.65 (m, 1H, aromatic), 8.76-8.78 (m, 1H,aromatic), 9.05 (s, 0.6H, aromatic), 9.09-9.14 (m, 1H, aromatic),8.37-8.39 (m, 1H, aromatic), 8.49 (s, 0.4H, aromatic).

Referential Example 13 Synthesis of Code Name GIF-0614

Sodium hydride (60%(w/w) oil mixture) (200 mg, 0.500 mmol) was added at0° C. to an N,N-dimethylformamide (DMF; 0.5 ml) solution ofN-[2-(1-piperidinyl)-5-(trifluoromethyl)phenyl]isonicotinamide (SRPIN-1,GIF-0340) (121 mg, 0.496 mmol), obtained as described in Referentialexample 1-3. The resulting mixture was stirred for one hour, and anN,N-dimethylformamide (DMF) solution of methyl iodide (0.8 M, 0.62 ml,0.496 mmol) was added thereto at 0° C. The resulting mixture was stirredfor three hours. Water was added to the mixture, and the resultingmixture was extracted three times with ethyl acetate. The obtainedorganic layer was washed with a saturated sodium chloride solution,dried over anhydrous sodium sulfate, filtered, and concentrated underreduced pressure. The residue was purified by silica gel columnchromatography (12 g, hexane/ethyl acetate=1/1). Thus,N-methyl-N-[2-(1-piperidinyl)-5-(trifluoromethyl)phenyl]isonicotinamide(GIF-0614) (65.9 mg, 51.5%) was yielded as a colorless solid.

The melting point, and results of TLC and ¹H NMR (CDCl₃, 400 MHz), areas follows: m.p. 119-121° C.; TLC R_(f) 0.36 (hexane/ethyl acetate=1/1);¹H NMR (CDCl₃, 400 MHz) δ 1.50-1.60 (m, 2H, CH₂), 1.60-1.70 (m, 2H,CH₂), 1.60-1.70 (m, 2H, CH₂), 2.39-2.41 (m, 2H, CH₂), 2.80-2.82 (m, 2H,CH₂), 3.20 (s, 3H, CH₃), 6.86 (d, 1H, J=8.3 Hz, aromatic), 7.15 (d, 2H,J=4.4 Hz, aromatic), 7.41 (d, 2H, J=8.3 Hz, aromatic), 7.48 (s, 1H,aromatic), 8.44 (d, 2H, J=4.4 Hz, aromatic).

Referential Example 14 Synthesis of Code Name GIF-0616

Lawesson's reagent (328 mg, 0.811 mmol, commercially available product)was added to a toluene (2.5 ml) solution ofN-[2-(1-piperidinyl)-5-(trifluoromethyl)phenyl]isonicotinamide (SRPIN-1,GIF-0340) (528 mg, 1.51 mmol), obtained as described in ReferentialExample 1-3, and the resulting mixture was stirred with refluxing at100° C. for 12 hours. The mixture was cooled to room temperature, andthen an aqueous solution of 2 M sodium hydroxide was added thereto toalkalify the solution. The mixture was reverse extracted three timeswith an aqueous solution of 12 M sodium hydroxide. 2 M hydrochloric acidwas added to the aqueous layer to acidify the solution. Then, theresulting mixture was extracted three times with ether. The obtainedorganic layer was washed with a saturated sodium chloride solution,dried over anhydrous sodium sulfate, filtered, and concentrated underreduced pressure. The residue was purified by silica gel columnchromatography (50 g, hexane/ethyl acetate=1/1). Thus,N-[2-(1-piperidinyl)-5-(trifluoromethyl)phenyl]isonicotinthioamide(GIF-0616) (186 mg, 33.7%) was yielded as a colorless solid.

The melting point, and results of TLC and ¹H NMR (CDCl₃, 400 MHz), areas follows: m.p. 108-109° C.; TLC R_(f) 0.27 (hexane/ethyl acetate=1/1);¹H NMR (CDCl₃, 400 MHz) δ 1.61-1.62 (m, 2H, CH₂), 1.68 (tt, 4H, J=5.0,5.0 Hz, 2CH₂), 2.87 (t, 4H, J=5.0 Hz, 2CH₂), 7.32 (d, 1H, J=7.8 Hz,aromatic), 7.51 (dd, 1H, J=1.6 Hz, aromatic), 7.71 (dd, 2H, J=1.6, 6.4Hz, aromatic), 8.76 (dd, 2H, J=1.6, 6.4 Hz, aromatic), 9.58 (d, 1H,J=1.6 Hz, aromatic), 10.5 (s, 1H, NH).

Referential Example 15 Synthesis of Code Name GIF-0341

Referential Example 15-1

Piperidine (660 μl, 6.66 mmol, commercially available product) was addedat room temperature to an N,N-dimethylformamide (DMF; 1 ml) solution of1,4-dichloro-2-nitrobenzene (390 mg, 2.03 mmol, commercially availableproduct). The resulting mixture was stirred for 18.5 hours. Water wasadded to the mixture, and the resulting mixture was extracted threetimes with ethyl acetate. The obtained organic layer was washed with asaturated sodium chloride solution, dried over anhydrous sodium sulfate,filtered, and concentrated under reduced pressure. The residue waspurified by silica gel column chromatography (30 g, hexane/ethylacetate=10/1). Thus, 1-(4-chloro-2-nitrophenyl)piperidine (471 mg,96.4%) was yielded as an orange-colored oily material.

TLC R_(f) 0.18 (hexane alone).

Referential Example 15-2

Concentrated hydrochloric acid (2.00 ml, 24.0 mmol) and anhydrous tindichloride (1.84 g, 9.70 mmol) were sequentially added at 0° C. to amethanol (10 ml) solution of 1-(4-chloro-2-nitrophenyl)piperidine (471mg, 1.95 mmol), obtained as described in Referential Example 15-1. Theresulting mixture was warmed to room temperature and stirred for 16hours. A saturated aqueous solution of sodium bicarbonate was added tothe mixture. The mixture was extracted three times with ethyl acetate.The obtained organic layer was washed with a saturated sodium chloridesolution, dried over anhydrous sodium sulfate, filtered, andconcentrated under reduced pressure. The residue was purified by silicagel column chromatography (30 g, hexane/ethyl acetate=9/1). Thus,5-chloro-2-(1-piperidinyl)aniline (388 mg, 94.3%) was yielded as acolorless oily material.

TLC R_(f) 0.26 (hexane/ethyl acetate=18/1).

Referential Example 15-3

Isonicotinoyl chloride hydrochloride (350 mg, 1.96 mmol, commerciallyavailable product), triethylamine (740 μl, 5.30 mmol), and a catalyticamount of 4-(dimethylamino)pyridine were sequentially added at roomtemperature to a dichloromethane (10 ml) solution of5-chloro-2-(1-piperidinyl)aniline (378 mg, 1.79 mmol), obtained asdescribed in Referential Example 15-2. The resulting mixture was stirredfor 19 hours. Water was added to the mixture, and the resulting mixturewas extracted three times with ethyl acetate. The obtained organic layerwas washed with a saturated sodium chloride solution, dried overanhydrous sodium sulfate, filtered, and concentrated under reducedpressure. The residue was purified by silica gel column chromatography(200 g, hexane/ethyl acetate=1/1). Thus,N-[5-chloro-2-(1-piperidinyl)phenyl]isonicotinamide (GIF-0341) (180 mg,31.8%) was yielded as a colorless solid.

The melting point, and results of TLC and ¹H NMR (CDCl₃, 400 MHz), areas follows: m.p. 141-143° C.; TLC R_(f) 0.32 (hexane/ethyl acetate=1/1);¹H NMR (CDCl₃, 400 MHz) δ 1.61-1.62 (m, 2H, CH₂), 1.76 (tt, 4H, J=5.0,5.0 Hz, 2CH₂), 2.82 (t, 4H, J=5.0 Hz, 2CH₂), 7.09 (dd, 1H, J=2.6, 8.8Hz, aromatic), 7.14 (d, 1H, J=8.8 Hz, aromatic), 7.75 (dd, 2H, J=1.6,4.4 Hz, aromatic), 8.60 (d, 1H, J=2.6 Hz, aromatic), 8.85 (dd, 2H,J=1.6, 4.4 Hz, aromatic), 9.66 (s, 1H, NH).

Referential Example 16 Synthesis of Code Name GIF-0342

Referential Example 16-1

Piperidine (660 μl, 6.66 mmol, commercially available product) was addedat room temperature to an N,N-dimethylformamide (DMF; 1 ml) solution of4-chloro-3-nitrotoluene (358 mg, 2.08 mmol, commercially availableproduct). The resulting mixture was stirred at 100° C. for 17 hours. Themixture was cooled to room temperature, and then water was addedthereto. The resulting mixture was extracted three times with ethylacetate. The obtained organic layer was washed with a saturated sodiumchloride solution, dried over anhydrous sodium sulfate, filtered, andconcentrated under reduced pressure. The residue was purified by silicagel column chromatography (50 g, hexane/ethyl acetate=50/1). Thus,1-(4-methyl-2-nitrophenyl)piperidine (212 mg, 46.2%) was yielded as acolorless oily material. TLC R_(f) 0.54 (hexane/ethyl acetate=10/1).

Referential Example 16-2

Concentrated hydrochloric acid (0.70 ml, 8.4 mmol) and anhydrous tindichloride (834 mg, 4.39 mmol) were sequentially added at 0° C. to amethanol (5 ml) solution of 1-(4-methyl-2-nitrophenyl)piperidine (212mg, 0.880 mmol), obtained as described in Referential Example 16-1. Theresulting mixture was warmed to room temperature and stirred for 16hours. A saturated aqueous solution of sodium bicarbonate was added tothe mixture. The mixture was extracted three times with ethyl acetate.The obtained organic layer was washed with a saturated sodium chloridesolution, dried over anhydrous sodium sulfate, filtered, andconcentrated under reduced pressure. The residue was purified by silicagel column chromatography (20 g, hexane/ethyl acetate=12/1). Thus,5-methyl-2-(1-piperidinyl)aniline (164 mg, 95.0%) was yielded as a paleyellow oily material.

TLC R_(f) 0.36 (hexane/ethyl acetate=10/1)

Referential Example 16-3

Isonicotinoyl chloride hydrochloride (172 mg, 0.966 mmol, commerciallyavailable product), triethylamine (340 μl, 2.44 mmol), and a catalyticamount of 4-(dimethylamino) pyridine were sequentially added at roomtemperature to a dichloromethane (5 ml) solution of5-methyl-2-(1-piperidinyl)aniline (155 mg, 0.815 mmol), obtained asdescribed in Referential Example 16-2. The resulting mixture was stirredfor 19 hours. Water was added to the mixture, and the resulting mixturewas extracted three times with ethyl acetate. The obtained organic layerwas washed with a saturated sodium chloride solution, dried overanhydrous sodium sulfate, filtered, and concentrated under reducedpressure. The residue was purified by silica gel column chromatography(10 g, hexane/ethyl acetate=1/1). Thus,N-[5-methyl-2-(1-piperidinyl)phenyl]isonicotinamide (GIF-0342) (69.5 mg,28.8%) was yielded as a colorless solid.

The melting point, and results of TLC and ¹H NMR (CDCl₃, 400 MHz), areas follows: m.p. 142-144° C.; TLC R_(f) 0.35 (hexane/ethyl acetate=1/1);¹H NMR (CDCl₃, 400 MHz) δ 1.62-1.70 (m, 2H, CH₂), 1.75 (tt, 4H, J=4.9,4.9 Hz, 2CH₂), 2.37 (s, 3H, CH₃), 2.82 (t, 4H, J=4.9 Hz, 2CH₂), 6.94(dd, 1H, J=1.6, 8.1 Hz, aromatic), 7.12 (d, 1H, J=8.1 Hz, aromatic),7.76 (dd, 2H, J=1.3, 4.5 Hz, aromatic), 8.38 (d, 1H, J=1.6 Hz,aromatic), 8.84 (dd, 2H, J=1.3, 4.5 Hz, aromatic), 9.75 (s, 1H, NH).

Referential Example 17 Synthesis of Code Name GIF-0348

Referential Example 17-1

Piperidine (320 μl, 3.23 mmol, commercially available product) was addedat room temperature to an N,N-dimethylformamide (DMF; 0.5 ml) solutionof 1,4-difluoro-2-nitrobenzene (225 mg, 1.41 mmol, commerciallyavailable product). The resulting mixture was stirred for two hours.Water was added to the mixture, and the resulting mixture was extractedthree times with ethyl acetate. The obtained organic layer was washedwith a saturated sodium chloride solution, dried over anhydrous sodiumsulfate, filtered, and concentrated under reduced pressure. The residuewas purified by silica gel column chromatography (20 g, hexane/ethylacetate=12/1). Thus, 1-(4-fluoro-2-nitrophenyl)piperidine (298 mg,94.2%) was yielded as an orange-colored oily material.

TLC R_(f) 0.46 (hexane/ethyl acetate=12/1).

Referential Example 17-2

Concentrated hydrochloric acid (1.20 ml, 14.4 mmol) and anhydrous tindichloride (1.22 g, 6.43 mmol) were sequentially added at 0° C. to amethanol (5 ml) solution of 1-(4-fluoro-2-nitrophenyl)piperidine (289mg, 1.28 mmol), obtained as described in Referential Example 17-1. Theresulting mixture was warmed to room temperature and stirred for 21hours. A saturated aqueous solution of sodium bicarbonate was added tothe mixture. The mixture was extracted three times with ethyl acetate.The obtained organic layer was washed with a saturated sodium chloridesolution, dried over anhydrous sodium sulfate, filtered, andconcentrated under reduced pressure. The residue was purified by silicagel column chromatography (25 g, hexane/ethyl acetate=12/1). Thus,5-fluoro-2-(1-piperidinyl)aniline (250 mg, quant.) was yielded as acolorless oily material.

TLC R_(f) 0.34 (hexane/ethyl acetate=16/1)

Referential Example 17-3

Isonicotinoyl chloride hydrochloride (454 mg, 2.55 mmol, commerciallyavailable product) and triethylamine (385 μl, 3.83 mmol) weresequentially added at 0° C. to a dichloromethane (10 ml) solution of5-fluoro-2-(1-piperidinyl)aniline (248 mg, 1.27 mmol), obtained asdescribed in Referential Example 17-2. The resulting mixture was warmedto room temperature and stirred for 17 hours. Water was added to themixture, and the resulting mixture was extracted three times with ethylacetate. The obtained organic layer was washed with a saturated sodiumchloride solution, dried over anhydrous sodium sulfate, filtered, andconcentrated under reduced pressure. The residue was purified by silicagel column chromatography (15 g, hexane/ethyl acetate=1.5/1). Thus,N-[5-fluoro-2-(1-piperidinyl)phenyl]isonicotinamide (GIF-0348) (257 mg,67.6%) was yielded as a colorless solid.

The melting point, and results of TLC and ¹H NMR (CDCl₃, 400 MHz), areas follows: m.p. 115-116° C.; TLC R_(f) 0.40 (hexane/ethyl acetate=1/1);¹H NMR (CDCl₃, 400 MHz) δ 1.62-1.69 (m, 2H, CH₂), 1.76 (bs, 4H, 2CH₂),2.82 (bs, 4H, 2CH₂), 6.81 (ddd, 1H, J=2.8, 8.8, 10.8 Hz, aromatic), 7.18(dd, 1H, J=5.6, 8.8 Hz, aromatic), 7.75 (dd, 2H, J=2.0, 4.4 Hz,aromatic), 8.34 (dd, 1H, J=2.8, 10.8 Hz, aromatic), 9.16 (dd, 2H, J=2.0,4.4 Hz, aromatic), 9.83 (s, 1H, NH).

Referential Example 18 Synthesis of Code Name GIF-0349

Referential Example 18-1

Piperidine (338 μl, 3.41 mmol, commercially available product) was addedat room temperature to an N,N-dimethylformamide (DMF; 0.5 ml) solutionof 2-fluoro-1-nitrobenzene (219 mg, 1.55 mmol, commercially availableproduct). The resulting mixture was stirred for two hours. Water wasadded to the mixture, and the resulting mixture was extracted threetimes with ethyl acetate. The obtained organic layer was washed with asaturated sodium chloride solution, dried over anhydrous sodium sulfate,filtered, and concentrated under reduced pressure. The residue waspurified by silica gel column chromatography (20 g, hexane/ethylacetate=20/1). Thus, 1-(2-nitrophenyl)piperidine (315 mg, 98.8%) wasyielded as a colorless oily material.

TLC R_(f) 0.53 (hexane/ethyl acetate=16/1).

Referential Example 18-2

Concentrated hydrochloric acid (1.50 ml, 18.0 mmol) and anhydrous tindichloride (1.45 g, 7.64 mmol) were sequentially added at 0° C. to amethanol (10 ml) solution of 1-(2-nitrophenyl)piperidine (315 mg, 1.52mmol) obtained as described in Referential Example 18-1. The resultingmixture was warmed to room temperature and stirred for 17 hours. Asaturated aqueous solution of sodium bicarbonate was added to themixture. The resulting mixture was extracted three times with ethylacetate. The obtained organic layer was washed with a saturated sodiumchloride solution, dried over anhydrous sodium sulfate, filtered, andconcentrated under reduced pressure. The residue was purified by silicagel column chromatography (20 g, hexane/ethyl acetate=15/1). Thus,2-(1-piperidinyl)aniline (238 mg, 88.8%) was yielded as a pale yellowoily material.

TLC R_(f) 0.19 (hexane/ethyl acetate=18/1)

Referential Example 18-3

Isonicotinoyl chloride hydrochloride (616 mg, 3.46 mmol, commerciallyavailable product) and triethylamine (800 μl, 5.73 mmol) weresequentially added at 0° C. to a dichloromethane (5 ml) solution of2-(1-piperidinyl)aniline (203 mg, 1.15 mmol), obtained as described inReferential Example 18-2. The resulting mixture was warmed to roomtemperature and stirred for two hours. Water was added to the mixture,and the resulting mixture was extracted three times with ethyl acetate.The obtained organic layer was washed with a saturated sodium chloridesolution, dried over anhydrous sodium sulfate, filtered, andconcentrated under reduced pressure. The residue was purified by silicagel column chromatography (20 g, hexane/ethyl acetate=1/1). Thus,N-[2-(1-piperidinyl)phenyl]isonicotinamide (GIF-0349) (259 mg, 80.0%)was yielded as a colorless solid.

The melting point, and results of TLC and ¹H NMR (CDCl₃, 400 MHz), areas follows: m.p. 111-113° C.; TLC R_(f) 0.35 (hexane/ethyl acetate=1/1);¹H NMR (CDCl₃, 400 MHz) δ 1.62-1.67 (m, 2H, CH₂), 1.76 (tt, 4H, J=4.8,4.8 Hz, 2CH₂), 2.85 (t, 4H, J=4.8 Hz, 2CH₂), 7.13 (td, 1H, J=1.6, 7.8Hz, aromatic), 7.21 (td, 1H, J=1.6, 7.8 Hz, aromatic), 7.24 (dd, 1H,J=1.6, 7.8 Hz, aromatic), 7.77 (dd, 2H, J=1.9, 4.4 Hz, aromatic), 8.53(dd, 1H, J=1.6, 7.8 Hz, aromatic), 8.84 (dd, 2H, J=1.9, 4.4 Hz,aromatic), 9.71 (s, 1H, NH).

Referential Example 19 Synthesis of Code Name GIF-0619

Referential Example 19-1

Indoline (402 μl, 3.59 mmol, commercially available product) andN,N-diisopropylethylamine (619 μl , 3.59 mmol) were sequentially addedat 0° C. to a N,N-dimethylformamide (DMF; 2 ml) solution of1-fluoro-2-nitro-4-(trifluoromethyl)benzene (498 mg, 2.38 mmol,commercially available product). The resulting mixture was warmed toroom temperature and stirred for one hour. The mixture was then heatedat 70° C. for 5.5 hours with stifling. The mixture was cooled to roomtemperature. Water was added to the mixture, and the resulting mixturewas extracted three times with ethyl acetate. The obtained organic layerwas washed with a saturated sodium chloride solution, dried overanhydrous sodium sulfate, filtered, and concentrated under reducedpressure. The residue was purified by silica gel column chromatography(30 g, hexane/ethyl acetate=20/1). Thus,1-(2-nitro-4-(trifluoromethyl)phenyl)indoline (730 mg, 99.4%) wasyielded as a deep red oily material.

TLC R_(f) 0.48 (hexane/ethyl acetate=6/1).

Referential Example 19-2

Concentrated hydrochloric acid (1.28 ml, 15.4 mmol) and anhydrous tindichloride (1.57 g, 8.30 mmol) were sequentially added at 0° C. to amethanol (7 ml) solution of1-(2-nitro-4-(trifluoromethyl)phenyl)indoline (730 mg, 2.37 mmol),obtained as described in Referential Example 19-1. The resulting mixturewas warmed to room temperature, and stirred for eight hours. A saturatedaqueous solution of sodium bicarbonate was added to the mixture. Theresulting mixture was extracted three times with ethyl acetate. Theobtained organic layer was washed with a saturated sodium chloridesolution, dried over anhydrous sodium sulfate, filtered, andconcentrated under reduced pressure. The residue was purified by silicagel column chromatography (50 g, hexane/ethyl acetate=10/1). Thus,1-[2-amino-4-(trifluoromethyl)phenyl]indoline (619 mg, 93.9%) wasyielded as a red-orange colored oily material.

TLC R_(f) 0.27 (hexane/ethyl acetate=6/1).

Referential Example 19-3

Isonicotinoyl chloride hydrochloride (669 mg, 3.76 mmol, commerciallyavailable product) and triethylamine (773 μl, 5.58 mmol) weresequentially added at 0° C. to a dichloromethane (5 ml) solution of1-[2-amino-4-(trifluoromethyl)phenyl]indoline (518 mg, 1.86 mmol),obtained as described in Referential Example 19-2. The resulting mixturewas warmed to room temperature and stirred for 2.5 hours. Water wasadded to the mixture, and the resulting mixture was extracted threetimes with ethyl acetate. The obtained organic layer was washed with asaturated sodium chloride solution, dried over anhydrous sodium sulfate,filtered, and concentrated under reduced pressure. The residue waspurified by silica gel column chromatography (50 g, hexane/ethylacetate=1/1). Thus,N-[2-(1-indolinyl)-5-(trifluoromethyl)phenyl]isonicotinamide (GIF-0619)(643 mg, 90.1%) was yielded as a colorless solid.

TLC R_(f) 0.32 (hexane/ethyl acetate=1/1).

Referential Example 20 Synthesis of Code Name GIF-0620

Referential Example 20-1

Isoindoline (679 μl, 5.98 mmol, commercially available product) wasadded at 0° C. to an N,N-dimethylformamide (DMF; 2 ml) solution of1-fluoro-2-nitro-4-(trifluoromethyl)benzene (508 mg, 2.43 mmol,commercially available product). The resulting mixture was warmed toroom temperature and stirred for two hours. Water was added to themixture, and the resulting mixture was extracted three times with ethylacetate. The obtained organic layer was washed with a saturated sodiumchloride solution, dried over anhydrous sodium sulfate, filtered, andconcentrated under reduced pressure. The residue was purified by silicagel column chromatography (30 g, hexane/ethyl acetate=15/1). Thus,2-[2-nitro-4-(trifluoromethyl)phenyl]isoindoline (749 mg, quant.) wasyielded as a yellow solid.

TLC R_(f) 0.42 (hexane/ethyl acetate=10/1).

Referential Example 20-2

Concentrated hydrochloric acid (1.13 ml, 13.5 mmol) and anhydrous tindichloride (1.38 g, 7.28 mmol) were sequentially added at 0° C. to amethanol (7 ml) solution of2-[2-nitro-4-(trifluoromethyl)phenyl]isoindoline (641 mg, 2.08 mmol),obtained as described in Referential Example 20-1. The resulting mixturewas warmed to room temperature and stirred for 8.5 hours. A saturatedaqueous solution of sodium bicarbonate was added to the mixture. Theresulting mixture was extracted three times with ethyl acetate. Theobtained organic layer was washed with a saturated sodium chloridesolution, dried over anhydrous sodium sulfate, filtered, andconcentrated under reduced pressure. The residue was purified by silicagel column chromatography (30 g, hexane/ethyl acetate=15/1). Thus,2-[2-amino-4-(trifluoromethyl)phenyl]isoindoline (225 mg, 38.9%) wasyielded as a red-orange colored oily material.

TLC R_(f) 0.38 (hexane/ethyl acetate=10/1).

Referential Example 20-3

Isonicotinoyl chloride hydrochloride (250 mg, 1.40 mmol, commerciallyavailable product) and triethylamine (287 μl, 2.07 mmol) weresequentially added at 0° C. to a dichloromethane (6 ml) solution of2-[2-amino-4-(trifluoromethyl)phenyl]isoindoline (193 mg, 0.694 mmol),obtained as described in Referential Example 20-2. The resulting mixturewas warmed to room temperature and stirred for three hours. Water wasadded to the mixture, and the resulting mixture was extracted threetimes with ethyl acetate. The obtained organic layer was washed with asaturated sodium chloride solution, dried over anhydrous sodium sulfate,filtered, and concentrated under reduced pressure. The residue waspurified by silica gel column chromatography (5 g, hexane/ethylacetate=1/1). Thus,N-[2-(2-isoindolinyl)-5-(trifluoromethyl)phenyl]isonicotinamide(GIF-0620) (266 mg, quant.) was yielded as a colorless solid.

TLC R_(f) 0.31 (hexane/ethyl acetate=1/1).

Referential Example 21 Synthesis of Code Name GIF-0621

Referential Example 21-1

1,2,3,4-tetrahydroisoquinoline (909 μl, 7.26 mmol, commerciallyavailable product) was added at 0° C. to an N,N-dimethylformamide (DMF;4 ml) solution of 1-fluoro-2-nitro-4-(trifluoromethyl)benzene (506 g,2.42 mmol, commercially available product). The resulting mixture waswarmed to room temperature and stirred for 3.5 hours. Water was added tothe mixture, and the resulting mixture was extracted three times withethyl acetate. The obtained organic layer was washed with a saturatedsodium chloride solution, dried over anhydrous sodium sulfate, filtered,and concentrated under reduced pressure. The residue was purified bysilica gel column chromatography (30 g, hexane/ethyl acetate=10/1).Thus,1,2,3,4-tetrahydro-2-[2-nitro-4-(trifluoromethyl)phenyl]isoquinoline(779 mg, 99.9%) was yielded as an orange-colored solid.

TLC R_(f) 0.54 (hexane/ethyl acetate=10/1).

Referential Example 21-2

Concentrated hydrochloric acid (1.11 ml, 13.3 mmol) and anhydrous tindichloride (1.35 g, 7.12 mmol) were sequentially added at 0° C. to amethanol (8 ml) solution of1,2,3,4-tetrahydro-2-[2-nitro-4-(trifluoromethyl)phenyl]isoquinoline(658 mg, 2.04 mmol), obtained as described in Referential Example 21-1.The resulting mixture was warmed to room temperature and stirred for 18hours. A saturated aqueous solution of sodium bicarbonate was added tothe mixture. The resulting mixture was extracted three times with ethylacetate. The obtained organic layer was washed with a saturated sodiumchloride solution, dried over anhydrous sodium sulfate, filtered, andconcentrated under reduced pressure. The residue was purified by silicagel column chromatography (30 g, hexane/ethyl acetate=20/1). Thus,2-[2-amino-4-(trifluoromethyl)phenyl]-1,2,3,4-tetrahydroisoquinoline(426 mg, 71.4%) was yielded as a red-orange colored oily material.

TLC R_(f) 0.36 (hexane/ethyl acetate=10/1).

Referential Example 21-3

Isonicotinoyl chloride hydrochloride (390 mg, 2.19 mmol, commerciallyavailable product) and triethylamine (449 μl, 3.24 mmol) weresequentially added at 0° C. to a dichloromethane (5 ml) solution of2-[2-amino-4-(trifluoromethyl)phenyl]-1,2,3,4-tetrahydroisoquinoline(315 mg, 1.08 mmol), obtained as described in Referential Example 21-2.The resulting mixture was warmed to room temperature and stirred forhalf an hour. Water was added to the mixture, and the resulting mixturewas extracted three times with dichloromethane. The obtained organiclayer was washed with a saturated sodium chloride solution, dried overanhydrous sodium sulfate, filtered, and concentrated under reducedpressure. The residue was purified by silica gel column chromatography(20 g, hexane/ethyl acetate=2/1). Thus,N-[2-(1,2,3,4-tetrahydroisoquinolin-2-yl)-5-(trifluoromethyl)phenyl]isonicotinamide(GIF-0621) (418 mg, 97.6%) was yielded as a colorless solid.

TLC R_(f) 0.52 (hexane/ethyl acetate=1/1).

Referential Example 22 Synthesis of Code Name GIF-0608

Isonicotinoyl chloride hydrochloride (670 mg, 3.76 mmol, commerciallyavailable product) and triethylamine (864 μl, 6.20 mmol) weresequentially added at 0° C. to a dichloromethane (5 ml) solution of3-(trifluoromethyl)aniline (208 mg, 1.29 mmol, commercially availableproduct). The resulting mixture was warmed to room temperature andstirred for 23 hours. Water was added to the mixture, and the resultingmixture was extracted three times with ethyl acetate. The obtainedorganic layer was washed with a saturated sodium chloride solution,dried over anhydrous sodium sulfate, filtered, and concentrated underreduced pressure. The residue was purified by recrystallization (ethylacetate). Thus, N-[3-(trifluoromethyl)phenyl]isonicotinamide (GIF-0608)(166 mg, 48.3%) was yielded as a colorless solid.

TLC R_(f) 0.26 (hexane/ethyl acetate=1/2).

Referential Example 23 Synthesis of Code Name GIF-0612

Isonicotinoyl chloride hydrochloride (427 mg, 2.39 mmol, commerciallyavailable product) and triethylamine (410 μl, 2.94 mmol) weresequentially added at 0° C. to a dichloromethane (5 ml) solution of2-bromo-5-(trifluoromethyl)aniline (480 mg, 2.00 mmol; commerciallyavailable product). The resulting mixture was warmed to room temperatureand stirred for 24 hours. Water was added to the mixture, and theresulting mixture was extracted three times with ethyl acetate. Theobtained organic layer was washed with a saturated sodium chloridesolution, dried over anhydrous sodium sulfate, filtered, andconcentrated under reduced pressure. The residue was purified byrecrystallization (ethyl acetate). Thus,N-[2-bromo-5-(trifluoromethyl)phenyl]isonicotinamide (GIF-0612) (308 mg,44.8%) was yielded as a colorless solid.

TLC R_(f) 0.46 (hexane/ethyl acetate=1/1).

Referential Example 24 Testing the Toxicity of SRPIN-1

Chromosomal screening tests were carried out using mammalian cells toevaluate SRPIN-1 abnormalities. The mutagenicity of SRPIN-1 wasevaluated using Chinese hamster CHL cells (Dainippon Pharma Co., Ltd)and with the inducibility of chromosome abnormality as an indicator. Thetests used the metabolic activation method (+S9 mix) with a shorttreatment (six hours of treatment, 18 hours of restoration), and, in theabsence of a metabolic activation system, used a continuous treatmentmethod (24 hours of treatment). CHL cells were cultured in MEM Earle(GIBCO BRL) containing 10% fetal calf serum (ICN Flow) under 5% CO₂ at37° C. Assays by the metabolic activation method used S9 fraction(Oriental Yeast Co Ltd.; A. T. Natarajan et al., Mutation Res., 37, pp83-90 (1976)), which had been prepared from the liver of Sprague-Dawleyrats (male, 7 weeks old; Charles River Laboratories Japan, Inc) to whichphenobarbital was administered intraperitoneally once a day for fourconsecutive days (30 mg/kg in the first administration, and 60 mg/kg inthe second to fourth administrations) and at the third administration,5,6-benzoflavone was given intraperitoneally at a dose of 80 mg/kg. 1 mlof the S9 mix used in this assay contained 4 μmol of HEPES buffer (pH7.4), 5 μmol of MgCl₂, 33 μmol of KCl, 5 μmol of G6P, 4 μmol of NADP,and 0.3 ml of S9 fraction.

In the short treatment method, CHL cells (about 4×10³ cells/nil) werecultured in 5 ml of culture medium using a 60 mm dish. After three daysof culture, a 1.33 ml aliquot of culture medium was taken from the dish,0.83 ml of S9 mix was added thereto, and 0.5 ml of test solution wasimmediately added at various concentrations (final concentrations: 5,1.58, or 0.5 mg/ml; dissolved in an aqueous solution of 0.5%carboxymethylcellulose sodium (CMC-Na)). The cells were incubated forsix hours, and then washed with PBS. 5 ml of fresh culture medium wasadded to replace the culture medium. The cells were further cultured for18 hours. An aqueous solution (0.5 ml) of 0.5% CMC-Na was used as anegative control, while dimethylnitrosamine (DMN; a final concentrationof 500 μg/ml) was used as a positive control; the procedure used was thesame as described above. In the method with continuous treatment, CHLcells (about 4×10³ cells/ ml) were cultured in 5 ml of culture medium ina 60 mm dish. After three days of culture, 0.5 ml of the culture mediumwas taken from the dish. 0.5 ml of test solutions at variousconcentrations (final concentrations: 5, 1.58, or 0.5 mg/ml) were added.The cells were incubated for 24 hours (about 1.5 cell cycles) withoutfurther treatment. An aqueous solution (0.5 ml) of 0.5% CMC—Na was usedas a negative control, while mitomycin C (MMC; a final concentration0.05 μg/ml) was used as a positive control; the procedure used was thesame as described above.

0.1 ml of 10 μg/ml colcemide was added two hours before the culture wasterminated, and the cells were harvested by treatment with a 0.25%trypsin solution. After hypotonic treatment using a 0.075 M potassiumchloride solution, the cells were fixed with a mixed solution ofmethanol and acetic acid (3:1). The cells were dried, and thenGiemsa-stained. For each dose, 50 metaphasic cells were spread out forobservation and the type and frequency of structural and numericalchromosomal abnormalities was determined. Structural abnormalities werecategorized into: chromatid breakage (ctb), chromatid exchange (cte),chromosome breakage (csb), chromosome exchange (cse), and miscellaneous(five or more abnormalities, fragmentations, pulverizations, and such).The frequency of occurrence was recorded for each category ofabnormality. If a cell had at least one of these abnormalities, it wasregarded as abnormal. When the frequency of cell abnormality was lessthan 5%, the testing substance was judged to be negative; when thefrequency was 5% or higher and less than 10%, the testing substance wasjudged to be pseudo-positive; and when the frequency was 10% or higher,the substance was judged to be positive. When the frequency ofoccurrence of cells with abnormal chromosomes was found to be 10% orhigher in a group treated with a test substance, the test substance wasconcluded to be a substance that induced chromosome abnormalities. Inthe positive control groups, where the cells were treated with 500 μg/mlDMN and 0.5 μg/ml MMC, the frequency of occurrence of cells withchromosomal abnormalities was 26 (52.0%) and 24 cells (48.0%)respectively. No abnormalities were found in the negative control. Thus,these tests were judged to be appropriately performed.

The test results for the SRPIN-1-treated groups showed that SRPIN-1 wasnegative for the increase in the number of cells with both structuraland numerical chromosomal abnormalities when evaluated by both the shortand continuous treatment methods. Based on the above results, it wasconcluded that the compound did not have the ability to inducechromosomal abnormalities in mammalian culture cells under theexperimental conditions of the present invention.

Referential Example 25 In Vivo Administration of SRPIN-1

The toxicity of SRPIN-1 was tested by single-dose oral administration torats. SRPIN-1 was orally administered at a dose of 125, 250, 500, 1000,or 2000 mg/kg (in a volume of 10 ml per kg) to Slc:SD rats (five weeksold; Japan SLC, Inc.) whose weight gain and general conditions werenormal during the acclimatization period. Each group included two malesand two females. The animals were fasted from the evening of the daybefore administration until about four hours after administration. Noneof the male and female rats died, and no changes were detectable intheir general condition for two days after administration. Then, SRPIN-1was likewise orally administered at a dose of 2000 mg/kg to five maleand five female Slc:SD rats (five weeks old). The rats were observed for14 days after administration, and based on visual examinations any toxicsymptoms were recorded along with their severity and timing, the timerequired for restoration, and the death date. The results showed thatnone of the male and female rats died, and no abnormalities weredetectable in their general condition.

Example 1A Phosphorylation of SR Proteins in Cells Infected with HIV

3 μg of HIVpNL4-3 genome (Adachi, A. et al., 1986, J. Virol. 59:284-289)was introduced into human Flp-In-293 cells derived from fetal kidney(R750-07; purchased from Invitrogen) using 9 μl of Genejuice (70967-4;purchased from Novagen), a gene transfer reagent. After four days thecells were lysed with 1 ml of SDS-PAGE sample buffer, and heat-denaturedat 95° C. for three minutes. The lysate was immediately transferred ontoice and used as a protein sample.

The protein sample was analyzed using Western blotting. The sample wasfractionated by SDS-PAGE using Laemmli buffer and gel with a gradient of4% to 20% at 40 mA for 45 minutes. Molecular weights were determinedusing Broad Range Pre-stained Marker (02525-35; Nacalai) as a molecularweight marker. Then, the sample was transferred onto PROTRANNitrocellulose Membrane (BA85; purchased from Schleicher & SchuellBioScience) by semi-dry blotting using TransBlot SD Cell (170-3940;purchased from Bio-Rad) at 160 mA for 60 minutes. After blotting, themembrane was washed with TBS for five minutes with shaking. Then, themembrane was blocked with BlockingOne (03953-95; purchased from Nacalai)at room temperature for one hour. The membrane was washed again withTBS, and incubated at 4° C. overnight with mouse monoclonal antibody 104(Mab104; hybridoma was purchased from ATCC), mouse anti-SC35 antibody(S4045; purchased from BDTransduction), and mouse anti-SF2 monoclonalantibody (AK103: a gift from Dr. Adrian Krainer; Hanamura, A. et al.,1998, RNA 4:430-444; Kojima, T. et al., 2001, J. Biol. Chem.276:32247-56), which each recognize phosphorylated SR proteins and werediluted with TBS.

The membrane was washed three times with TBS at room temperature for tenminutes with shaking. Then, HRP-labeled sheep anti-mouse IgG antibody(NA9310; purchased from Amersham) was diluted with TBS, and the membranewas incubated with this secondary antibody at room temperature for onehour. The membrane was washed three times with TBS at room temperaturefor ten minutes with shaking. Then, the detection was carried out bychemical luminescence using ECL Detection Reagents (RPN2105; purchasedfrom Amersham) and images were photographed using a LAS 1000CCD camera(LAS 1000; Fuji Film). The results are shown in FIG. 1A.

The results showed that when the cells were infected with HIVpNL4-3,Western analysis using Mab104, SC35, and SF2 antibodies could not detectany signals. Thus, it was revealed that not only was SR proteindephosphorylated, but endogenous SR proteins, such as SC35 and SF2 werealso degraded.

Example 1B Degradation of SR Proteins

Using Genejuice, 1 μg each of the plasmids for the SRp75, SRp55, andSRp40 genes fused with HA tag (HA-SRp75, HA-SRp55, and HA-SRp40; giftsfrom Dr. Woan-Yuh TARN), were introduced into Flp-In-293 cells derivedfrom human fetal kidney. After 36 hours, MG132 (474790; purchased fromCalbiochem), a ubiquitin proteasome inhibitor, was added at a finalconcentration of 10 μM. After ten hours, the cells were lysed withSDS-PAGE sample buffer and heat-denatured. The resulting lysate was usedas a protein sample. Using the same procedures as described above, thesample was fractionated by SDS-PAGE, and analyzed by Western blottingusing a rabbit anti-HA antibody (H1803; purchased from Santa Cruz) as aprimary antibody and a donkey anti-rabbit IgG antibody (NA9340;purchased from Amersham) as a secondary antibody. The results are shownin FIG. 1B.

The cells treated with MG132 gave a stronger signal than the control,showing that MG132 inhibited degradation of SR75, SR55, and SR40proteins. In addition, similar results were obtained for other SRproteins (data not shown). Using MG132, it thus was revealed that SRproteins were degraded by ubiquitin proteasome.

Example 2A Phosphorylation of SR Proteins in Cells Stably ExpressingSRPK2

A single copy of the mouse SRPK2 gene was introduced into Flp-In-293cells at the Flp-In site to establish multiple cell lines stablyexpressing SRPK2. The parent cell line Flp-In-293 was used as a mock inthe analysis, and for use in the experiments SRPK2-2 was selected fromthe multiple established cell lines stably expressing SRPK2. pNL4-3 wasintroduced into these two cells. After four days, the dynamics ofendogenous SR protein during HIV infection were investigated usingWestern analysis.

Western analysis was carried out the same way as in FIG. 1A. When theHIVpNL4-3 genome was introduced into SRPK2-2 cells, Mab104 antibodydetected signals at positions corresponding to SRp35, SRp40, SRp55, andSRp75. The SR domains recognizable by Mab104 were found to bephosphorylated.

Furthermore, Western analysis using SC35 and SF2 antibodies revealedthat SC35 and SF2 signals were observed in SRPK2-2 cells introduced withthe HIVpNL4-3 genome. The results are shown in FIG. 2A.

These results showed that SR proteins are generally degraded upon HIVinfection, but in cells stably expressing SRPK2, the SR proteins remainphosphorylated and are stabilized as a result. This suggests that SRprotein is phosphorylated and that protein degradation via ubiquitinproteasome does not take place in cells stably expressing SRPK2.

Example 2B Existence of SR Proteins in Cells Stably Expressing SRPK2

1 μg of HIVpNL4-3 genome and 1 μg each of the plasmids for the SRp75,SRp55, and SRp40 genes fused with HA tag (HA-SRp75, HA-SRp55, HA-SRp40;gifts from Dr. Woan-Yuh TARN) were introduced into the Flp-In-293 cellsstably expressing SRPK2, where one copy of the SRPK2 gene had beenintroduced at the Flp-In site (SRPK2-2 cells) and the parental cell lineFlp-In-293 (mock) using Genejuice. The samples were collected after 36hours and analyzed by Western blotting. The results are shown in FIG.2B. According to these results, upon HIV infection of the Flp-In-293cells, the anti-HA antibody signal weakened or disappeared for not onlySC35 and SF2, but also for SRp75, SRp55, and SRp40. In the SRPK2-2cells, the signals for SRp75, SRp55, and SRp40 as well as SC35 and SF2were detectable, although impaired as compared with the control.

These results suggest that SR protein degradation is enhanced upon HIVinfection, but that SR protein phosphorylation in SRPK2-2 cellsstabilizes the SR proteins.

Example 2C Quantifying the Produced HIV

In the experiment in Example 2A (FIG. 2A), the culture supernatant wascollected and the HIV produced was quantified. After gene transfer, theculture supernatant was collected and the amount of HIV capsid protein‘p24’ comprised in the culture supernatant was measured using theLumipulse ELISA system (Fujirebio). The results are shown in FIG. 2C.These results showed that the SRPK2-2 cells produced 2.3 times more HIVthan the mock culture supernatant.

These findings revealed that SR proteins are dephosphorylated inresponse to HIV infection, but that if the SR proteins remainphosphorylated, the regulatory mechanism of SR proteins in response toinfection does not work, and thus HIV production is enhanced.

This suggests that the dephosphorylation of SR proteins functions as ahost defense mechanism in response to HIV infection.

Example 3A Evaluation of SR Proteins Contributing to In Vivo HIVProduction

In the process of HIV gene expression, HIV is transcribed, processed,and translated using host-derived factors. In particular, it has beenspeculated that the Tat and Rev of HIV have split exons and thus an mRNAsplicing reaction is essential for gene expression.

As shown in Examples 1-2 (FIGS. 1-2), a host defense mechanism isactivated upon HIV infection, and SR proteins are degraded as a result.However, there is no information about HIV's in vivo splicing reactions,nor the SR protein contribution to these reactions. In fact there aremany types of SR proteins in cells, and thus expression plasmids (0.5μg) for such SR proteins were introduced along with the HIVpNL4-3 genome(1.0 μg), and their effects were evaluated. The results are shown inFIG. 3A.

Each of the expression plasmids for mock, SC35, SF2, SRp40, SRp55, orSRp75 were introduced into Flp-In-293 cells. After 36 hours, the culturesupernatants were collected and the amount of HIVp24 was determinedusing the Lumipulse ELISA system.

According to the results, more HIVp24 was produced for SRp40 and SRp75than for the mock. Thus, SRp40 and SRp75 were found to have the effectof enhancing HIV production.

Example 3B Evaluation of the Effect of Using hnRNPA1 on In Vivo HIVProduction

As shown in FIG. 3B, in combination with HIVpNL4-3 genome (0.5 μg), afixed amount (500 ng) of expression plasmid for SRp40 or SRp75 andincreasing amounts of an expression plasmid for hnRNPA1 were introducedinto Flp-In-293 cells. After 36 hours, the culture supernatant wascollected and the amount of HIVp24 was determined using the LumipulseELISA system.

The results showed that the amount of HIVp24 determined by the LumipulseELISA system decreased depending on the dose of hnRNPA1. Specifically,hnRNPA1 suppresses HIV production, acting in competition with SRp40 andSRp75.

This suggests that HIV gene expression is regulated by splicingreactions in cells. Actually, since hnRNPA1 co-exists with SRp40 andSRp75 in cells, it is thought that HIV infection-induced degradation ofSR proteins in cells functions as a defense mechanism by allowinghnRNPA1 to dominate in cells and thus suppressing HIV gene expression.

Example 4A Search for SRPK Inhibitors of SR Protein Phosphorylation inCells

Inhibitors that competitively bind to the ATP binding site shared by thekinases were sought. One hit compound in the results of screening wasfound to be commercially available from Maybridge (molecularweight=349.35; CAS Registry No. 218156-96-8). However, no informationabout the inhibition of kinase has been previously disclosed. Thepresent inventors named the compound “SRPIN-1” (SRPk Inhibitor-1).

Example 4B Evaluation of the Inhibition of SRPK1 PhosphorylationActivity by SRPIN-1

An RS peptide (NH2-RSPSYGRSRSRSRSRSRSRSRSNSRSRSY-OH; SEQ ID NO: 5)corresponding to the RS domain of SF2 was synthesized. The peptide wasdissolved to a concentration of 1 mg/ml in 10 mM Tris-HCl (pH 7.5).SRPIN-1 (final concentration: 0.1, 0.3, 1.0, 3.0, or 10.0 μM) wasincubated with 1 μg of purified recombinant SRPK1 protein, which hadbeen expressed in E. coli, in a reaction buffer (250 μM MgCl₂, 0.25 mMATP, 1 mCi of [γ-³²P] ATP) in a 30° C. water bath for ten minutes. Theamounts of SRPK1 and RS peptide for the kinase activity assay, and theconditions for reaction time, were tested in advance and selected forreaction linearity.

SRPK1 and RS peptide were incubated together for ten minutes, then thereaction solution was dropped onto a P81 phosphocellulose membrane (P81;Whatman) and the membrane was washed with 5% phosphoric acid solution.After washing, ³²P radioactivity on the P81 membrane was determinedusing a liquid scintillation counter. The results are shown in FIG. 4B.

The results showed that the IC50 of SRPIN-1 for SRPK1 was about 400 nM.When tested using the same technique, CLK1, CLK2, CLK3, CLK4, SRPK2,PRP4, PKA, and PKC did not exhibit an inhibitory effect, even at thefinal concentration of 10 μM. It is thus safe to conclude that SRPIN-1is an SRPK1-specific inhibitor.

Example 4C Evaluation of In Vivo Inhibition of SR ProteinPhosphorylation by SRPIN-1 and the Accompanying Induction of SR ProteinDegradation

HA-SRp75 plasmid (1.0 μg) was introduced into Flp-In-293 cells. After 36hours, MG132 (final concentration: 10 μM) and SRPIN-1 (10, 20, or 50 μM)were added, and the cells were incubated for 15 hours. Then, the cellswere lysed with SDS-PAGE sample buffer. The lysate was used as a proteinsample. The sample was fractionated by SDS-PAGE and analyzed by Westernblotting using the anti-HA antibody. In addition, as a control forprotein amount, Western analysis was carried out using anti-beta actinantibody. The results are shown in FIG. 4C.

The result showed that the HA antibody signal weakened depending on theconcentration of SRPIN-1. This suggests that the endogenous SRPK1activity was inhibited in an SRPIN-1 dependent manner, and as a resultSRp75 protein was degraded.

This finding shows that the inhibition of SRPK1 by SRPIN-1 can result inthe inhibition of in vivo SR protein phosphorylation, labilizing SRprotein as a result, and thus enhancing protein degradation.

Example 4D Evaluation of the Inhibition of HIV Infection by AddingSRPIN-1

An infection experiment was carried out by adding HIV virions, whichwere prepared in 293T cells, to MT-4 cells. First, a prepared viralliquid and SRPIN-1 (final concentration: 0.5, 10, or 20 μM) weresimultaneously added to the MT-4 cells. The cells were incubated at 37°C. under 5% CO₂ for two hours, then centrifuged, and the culture mediumwas exchanged for fresh medium. Then, the culture supernatant wascollected after 48 hours, and the amount of HIVp24 was determined by theLumipulse ELISA system. The results are shown in FIG. 4D.

The result showed that the amount of HIVp24 as determined by theLumipulse ELISA system decreased in an SRPIN-1 concentration-dependentmanner. This suggests that SRPIN-1 can inhibit HIV production in aconcentration-dependent manner.

Example 5 Inhibition of SRPK1 or SRPK2 Phosphorylation Activity UsingSRPIN-1 Analogs

The same procedure as described in Example 4B was used to determinewhether SRPIN-1 analogs had the activity of inhibiting thephosphorylation activity of SRPK1 and SRPK2. Each SRPIN-1 analog (10 μM;in DMSO) was incubated with 1 μg of purified recombinant SRPK1 or SRPK2protein, which was expressed in E. coli, in a reaction buffer (400 μMHEPES (pH 7.5), 100 μM MgCl₂, 200 μM ATP, 1 mCi [γ-³²P] ATP, and 1 mg/mlRS peptide (SEQ ID NO: 5)) in a 30° C. water bath for 20 minutes.

After RS peptide was incubated with SRPK1 or SRPK2 for 20 minutes, thereaction solution was dropped onto P81 phosphocellulose membrane (P81;Whatman) and the membrane was washed three times for ten minutes with 5%phosphoric acid solution. After washing, ³²P radioactivity on the P81membrane was determined using a liquid scintillation counter. As shownin FIG. 5A, each SRPIN-1 analog exerted an inhibitory effect on thephosphorylation activity of SRPK1 and/or SRPK2. In particular, thecompounds of Compound Nos. 340 (SRPIN-1) to 348, 612, 613, 615, 618,619, 621, 624, and 625 were found to exhibit a strong inhibitory effecton SRPK1 or SRPK2.

Then, each SRPIN-1 analog was tested for its effect in suppressing HIVreplication. An infection experiment was carried out by adding HIVvirions, which were prepared in 293T cells, to MT-4 cells (JCRB No.JCRB0135). First, a prepared viral liquid and an SRPIN-1 analog (finalconcentration: 5, 10, or 20 μM) were simultaneously added to the MT-4cells. The cells were incubated at 37° C. under 5% CO₂ for two hours,then centrifuged, and the culture medium was exchanged for fresh medium.The culture supernatant was then collected after 48 hours, and theamount of HIVp24 was determined by the Lumipulse ELISA system. Theresults showed that each SRPIN-1 analog listed in FIG. 5B had theactivity of inhibiting HIV replication. In particular, the compounds ofCompound Nos. 340, 341, 342, 343, 345, 347, 348, 608, 613, 615, 616,618, 619, 620, 622, 623, 624, 625, and 626 were found to have strongeffects in suppressing HIV propagation. Furthermore, as shown in FIG.5C, the compounds were also found to have the effect of suppressing HIVpropagation in experiments using other cells (Jurkat).

Example 6 Suppressing Effect on Sindbis Virus Propagation

5 μl of sindbis virus (4.7×10⁷ PFU/ml) was added to Vero cells(JCRB0111) and the cells were cultured for 24 hours. The culturesupernatant was collected as stock virus, diluted to 10² to 10⁷ PFU,then added to BHK21 C13 cells (JCRB9020). SRPIN-1 was also added at thesame time (final concentration: 5, 10, 20, or 40 μM). After one hour ofinfection at room temperature, a medium comprising 1% methylcellulose(SIGMA M0512-100G) was added, and the cells were gently cultured at 37°C. under 5% CO₂ for 48 hours. Cell morphology was observed under a phasecontrast microscope, and the number of plaques formed by cell deathcaused by sindbis virus infection was counted (plaque assay) tocalculate the PFU/ml.

FIG. 6A shows phase contrast microscopic images of cells 20 hours aftervirus infection. Marked cell damage caused by sindbis virus propagationwas found in those cells not treated with SRPIN-1 (“+SIN, control” inthis Figure), while cell damage was dramatically suppressed byadministering SRPIN-1 (40 μM) (“+SIN, 40 μM (#340)” in this Figure). Theplaque assay results also revealed that a 5 μM or higher concentrationof SRPIN-1 significantly suppressed the propagation of sindbis virus ina concentration-dependent manner (FIG. 6B).

Example 7 Suppressing Effect on Cytomegalovirus Propagation

Cytomegalovirus (1×10⁴ PFU/ml) and SRPIN-1 or an analog thereof(Compound No. 340 or 349; final concentration: 20 or 40 μM) weresimultaneously added to HFL1 cells (IF050074). The HFL1 cells infectedwith cytomegalovirus were observed under a phase contrast microscopeseven days after infection. As shown in FIG. 7, morphological changescharacteristic of cytomegalovirus infection and cell death were foundwith high frequency in control group HFL1 cells (1 and 2 in thisFigure), to which no SRPIN-1 was added. In contrast, when SRPIN-1 (20μM) was added to the HFL1 cells, neither abnormal morphological changesnor cell death were detectable (3 in this Figure), despite thecytomegalovirus infection. Partial morphological changes thought to beinduced by SRPIN-1 were detectable in HFL1 cells to which SRPIN-1 wasadded at a higher concentration (40 μM). Further, addition of an SRPIN-1analog compound (Compound No. 349) at 20 or 40 μM to HFL1 cells alsosuppressed the abnormal morphological changes and cell death caused bycytomegalovirus infection (5 and 6 in this Figure). Thus, it wasdemonstrated that under these assay conditions SRPIN-1 and its analogcompounds could suppress the changes in cell morphology and cell deathcaused by cytomegalovirus infection.

Example 8 Suppressing Effect on SARS Virus Propagation

Vero cells (JCRB0111) were infected with SARS virus (FFM-1) (Yamamoto,N. et al., Biochem Biophys Res Commun. 318, 719-725 (2004)), andsimultaneously SRPIN-1 or an analog thereof (final concentration: 5, 10,20, or 40 μM) was added thereto. After two hours of infection at roomtemperature, D-MEM containing 1% methylcellulose (SIGMA M0512-100G) wasadded and the cells were cultured at 37° C. under 5% CO₂ for 48hours_(.) The number of plaques formed by cell death caused by SARSvirus infection was counted (plaque assay) to calculate PFU/ml. As shownin FIG. 8A, 40 μM SRPIN-1 and an analog compound thereof (Compound No.349) significantly suppressed SARS virus propagation. The viralpropagation-suppressing effect of SRPIN-1 was stronger than that of theanalog compound (Compound No. 349). In addition, as seen in FIG. 8B,SRPIN-1 was found to suppress SARS virus propagation in aconcentration-dependent manner within the concentration range of 1 to 40μM.

Industrial Applicability

The present invention revealed that SRPIN-1 (SR protein phosphorylationinhibitor 1) and analogs thereof have the activity of inhibiting SRPKkinases. When phosphorylated by SRPKs, SR proteins are stable in cells.However, SR proteins were found to be degraded via theubiquitin-proteasome pathway when SR protein phosphorylation wasinhibited by using SRPK inhibitors to inhibit SRPK enzymatic activity.Thus, the SRPK inhibitors were added to inhibit SRPK in HIV infectionexperiments, and were found to have the antiviral activity ofsuppressing viral replication.

The present invention is also beneficial in that it provides antiviralagents that control the activity of SR proteins, and thus by the samemechanism are effective against a broad range of viruses.

The invention claimed is:
 1. A compound represented by the followingformula (I):

or a pharmaceutically acceptable salt or hydrate thereof; wherein R¹represents a halogen atom; R² represents a hydrogen atom; R³ representsa nitrogen-containing heterocycle which may have a substituent; R⁴represents a hydrogen atom; Q represents —C(S)—; and W represents agroup represented by the following formula (II):

wherein R⁵ and R⁶, together with the adjacent nitrogen atom, form aheterocycle which may have a substituent.
 2. A pharmaceuticalcomposition comprising the compound according to claim 1 and apharmaceutically acceptable excipient.
 3. The compound of claim 1 or apharmaceutically acceptable salt or hydrate thereof, wherein R¹represents a fluorine atom; R³ represents a 4-pyridyl group; and Wrepresents a piperidinyl group.
 4. The compound of claim 1, wherein thecompound is represented by the following formula (III):

or a pharmaceutically acceptable salt or hydrate thereof.
 5. Apharmaceutical composition comprising the compound according to claim 3and a pharmaceutically acceptable excipient.
 6. A pharmaceuticalcomposition comprising the compound according to claim 4 and apharmaceutically acceptable excipient.